WO1997010362A1

WO1997010362A1 - Methods of using nucleotide integrase for cleaving dna and attaching nucleic acids

Info

Publication number: WO1997010362A1
Application number: PCT/US1996/014609
Authority: WO
Inventors: Alan M. Lambowitz; Steven Zimmerly; Jian Yang; Huatao Guo
Original assignee: The Ohio State Research Foundation
Priority date: 1995-09-12
Filing date: 1996-09-11
Publication date: 1997-03-20
Also published as: DE69636835D1; DE69636835T2; US5698421A; EP0851940B1; EP0851940A4; JPH11512291A; CA2229075C; US5869634A; EP0851940A1; AU715563B2; CA2229075A1; AU6974496A

Abstract

The present invention provides new methods, employing a nucleotide integrase, for cleaving double-stranded and single stranded DNA substrates at specific sites and for attaching nucleic acid molecules to the cleaved DNA substrates. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA and to concomitantly attach a nucleic acid molecule to the cleaved strand. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach a nucleic acid molecule to one strand of the DNA substrate. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach an RNA molecule to one strand of the substrate and for attaching a cDNA to the other strand of the substrate. Another method cleaves single stranded DNA with the concomitant insertion of a nucleic acid molecule at the cleavage point. The nucleotide integrase comprises an RNP particle which comprises a group II intron RNA bound to a group II intron encoded protein. The present invention also relates to purified and reconstituted RNP particles and reconstituted RNP that cleave DNA substrates.

Description

METHODS OF USING NUCLEOTIDE INTEGRASE FOR CLEAVING DNA AND ATTACHING NUCLEIC ACIDS

BACKGROUND In recent years, a number of methods and biomolecules have been developed for manipulating DNA. Some of these biomolecules are used to cut or cleave DNA, which in some instances renders the substrate DNA nonfunctional. Other biomolecules are used to facilitate insertion of new pieces of nucleic acid into the cleavage site of the DNA substrate. The insertion of new segments of nucleic acid into the cleavage sites of the DNA substrate changes the characteristics of the RNA or protein molecules encoded by the substrate DNA molecules. Accordingly, the biomolecules which catalyze the cleavage of DNA substrates or the insertion of new nucleic acid molecules into the DNA substrates are useful tools for genetic engineering, for analytical studies and for diagnostic studies. One such molecule used for cleaving DNA substrates is the restriction endonuclease.

Restriction endonucleases are enzymatic proteins that cleave double-stranded DNA. Such endonucleases recognize specific nucleotide sequences in double-stranded DNA, and cleave both strands within or near the specific recognition site. Such specificity renders the restriction endonucleases important tools in the controlled fragmentation of double-stranded DNA. Restriction endonucleases are also useful analytical tools for determining whether certain sequences are present in substrate DNA and in genomic sequencing studies.

However, restriction endonucleases only cleave DNA substrates; they do not insert new nucleic acid molecules into the cleaved DNA substrate. Accordingly, another biomolecule is needed to insert new pieces of DNA or RNA into the double-stranded DNA.

Ribozymes are catalytic RNA molecules that cleave RNA and, in certain circumstances, that insert new pieces of RNA into the cleavage site of the RNA substrate. Unfortunately, ribozymes have not been particularly useful for cleaving DNA. Ribozymes cut only single- stranded DNA and only under extreme conditions of elevated temperatures and high concentrations of magnesium. The ribozymes have not been found to cleave double-stranded DNA. Ribozymes can be used to cleave double-stranded DNA only after the DNA is denatured and separated into two pieces of single stranded DNA. Accordingly, it would be desirable to have a tool that cleaves double stranded DNA at specific sites and simultaneously inserts a new nucleic acid molecule into the cleavage site of the double-stranded DNA.

SUMMARY OF THE INVENTION The present invention provides new methods, employing a nucleotide integrase, for cleaving double stranded and single stranded DNA substrates at specific sites and for inserting nucleic ac d molecules into the cleaved DNA substrate. Thus, the nucleotide integrase is a useful tool, particularly for genome mapping and for genetic engineering. One method uses a nucleotide integrase to cleave one strand of double-stranded DNA at a specific site and to concomitantly attach a nucleic acid molecule, which comprises an RNA molecule, to the cleaved strand at the cleavage site. Another method uses a nucleotide integrase for cleaving both strands of double-stranded DNA and for attaching a nucleic acid molecule, which comprises an RNA molecule, to one strand of the DNA substrate. Preferably, the nucleic acid molecule is fully integrated into the cleavage site. Another method uses a nucleotide integrase for cleaving both strands of double-stranded DNA and for attaching an RNA molecule to one strand of the DNA substrate at the cleavage site and for attaching a cDNA to the other strand of the

DNA substrate at the cleavage site. Preferably, the RNA molecule is fully integrated into the cleavage site of the one strand. Another method provides for cleavage of single stranded DNA with the concomitant insertion of a nucleic acid at the cleavage point. The nucleotide integrase comprises a group II intron RNA and a group II intron encoded protein, which is bound to the group II intron RNA The nucleotide integrase includes at least two forms a ribonucleoprotem particle, also referred to herein as "RNP particle" , and a reconstituted particle. The RNP particle comprises an excised group II intron RNA and a group II intron-encoded protein, bound to the excised group II intron RNA. The excised group II intron RNA of the RNP particle has a hybridizing region which is complementary to a recognition site on the substrate DNA. Preferably,, the group II intron RNA has six domains. The group II intron-encoded protein of the RNP particle preferably has an X domain and a Zn domain. The X domain of the protein has a maturase activity. The Zn domain of the protein has Zn^2* fmger-like motifs.

The reconstituted particle is a synthetic particle that is purified from a reconstituted RNP particle preparation. The reconstituted RNP particle preparation is made by combining a group II mtron RNA-protem complex, also referred to as the "RNA-protem complex" and an exogenous nucleic acid, preferably an excised group II intron RNA, hereinafter also referred to as "exogenous RNA" The RNA- protem complex contains a group II mtron-encoded protein that is associated with RNA that is free of the excised group II intron RNA having a sequence which encodes the group II encoded protein. The intron-encoded protein has an X domain and a Zn domain. The exogenous RNA has a hybridizing region which is complementary to a recognition site on the substrate DNA. Preferably, the exogenous RNA has six domains.

The present invention also relates to certain embodiments of the nucleotide integrase.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of the domains of the proteins which are encoded by the open reading frames of the group II intron 2 of the S . cerevisiae mitochondrial COX1 gene, the group II intron 2 of the M. polymorpha mitochondrial COX1 gene, and the group II intron 1 of the N. tabacum chloroplast trnK gene; Figure 2 is a chart which depicts the locations of the mutations in the group II intron encoded proteins that are present in the nucleotide integrases of examples 4-9 and the RNP particles of comparative examples B-D;

Figure 3 is a diagram of the plasmid map of plasmid pJVM4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new methods that employ a nucleotide integrase for manipulating a DNA substrate. The methods enable double-stranded DNA substrates to be cleaved at specific sites and nucleic acid molecules to be inserted into the cleaved DNA substrate. One method uses a nucleotide integrase to cleave one strand of double-stranded DNA at a specific site and to concomitantly attach a nucleic acid molecule, which comprises an RNA molecule, to the cleaved strand at the cleavage point. Another method uses a nucleotide integrase for cleaving both strands of double-stranded DNA and for attaching a nucleic acid molecule, preferably an RNA molecule, to one strand of the DNA substrate at the cleavage point. Another method uses a nucleotide integrase to cleave both strands of double-stranded DNA and to attach a nucleic acid molecule, preferably an RNA molecule, to one strand of the DNA substrate and then to attach a cDNA molecule to the other strand of the DNA substrate at the cleavage site. The nucleotide inteσrase

The nucleotide integrase comprises a group II intron encoded RNA and a group II intron encoded protein which protein is bound to the RNA. The group II introns comprise a specific type of intron which is present in the DNA of bacteria and in the DNA of organelles, particularly the mitochondria of fungi, yeast and plants and the chloroplast of plants. The group II intron RNA molecules, that is, the RNA molecules which are encoded by the group II introns, share a similar secondary and tertiary structure. The group II intron RNA molecules typically have six domains. Domain IV of the group II tron RNA contains the nucleotide sequence which encodes the "group II intron encoded protein." "Excised group II intron RNA," as used herein, refers to the RNA that is either an n vitro or vivo transcript of the DNA of the group II intron and that lacks flanking exon sequences "Group II intron encoded protein" as used herein, is a protein encoded by a group II mtron. The domains of three representative group II mtron-encoded protems are depicted in Figure 1. The nucleotide integrase includes RNP particles isolated from wild type or mutant organisms and reconstituted RNP particles synthesized from exogenous RNA and a particle preparation which lacks the exogenous RNA. The RNP particle The RNP particle is used in an RNP particle preparation which, while isolated from organelles, still contains substantial amounts of ribosomes; or the RNP particle is used as a purified RNP particle which is substantially free of ribosomes.

The RNP particle and RNP particle preparation cleave both strands of the double-stranded DNA, catalyze the attachment of the excised, group II mtron RNA molecule to one of the strands of the substrate DNA and catalyze the formation of a cDNA molecule on the other strand of the cleaved double-stranded DNA substrate.

The RNP particle comprises an excised group II intron RNA and a group II intron-encoded protein, which is bound to the excised group II intron RNA. The excised group II intron RNA of the RNP particle has at least one hybridizing region, which will hybridize a complementary recognition site on the substrate DNA. The hybridizing region has a nucleotide sequence, referred to hereinafter as the EBS sequence, which is complementary to the sequence, referred to hereinafter as the IBS sequence, of the recognition site of the intended substrate DNA The group II mtron-encoded protein of the RNP particle preferably has an X domain and a Zn domain. More preferably, the group II intron-encoded protein further comprises a reverse transcriptase domain. The excised group II mtron RNA is obtained from wild type organisms, or mutated organisms, by m vitro transcription, or by in vitro transcription and splicing from the transcript of a modified or unmodified group II mtron. Nucleotide mtegrases in which the group II intron RNA has nucleotide base changes in the hybridizing region, as compared to the wild type, typically have altered specificity for the substrate DNA of the nucleotide integrase The group II mtron RNA also includes modified group II intron RNA molecules that have nucleotide base changes or additional nucleotides m the internal loop regions of the group II intron RNA, preferably the internal loop region of domain IV.

The group II intron-encoded protein preferably has an X domain and a Zn domain. The group II intron-encoded protein includes proteins isolated from wild type organisms or from mutant organisms . In addition to the conventional group II intron encoded proteins, other proteins suitable as components of the nucleotide integrase include modified group II intron encoded proteins which have additional amino acids at the N terminus, or C terminus, or alterations in the internal regions of the protein. Preferably, the group II-intron encoded protein has a reverse transcriptase domain. The Reconstituted particle

The reconstituted particle is used in a reconstituted RNP particle preparation which, while isolated from organelles, still contains substantial amounts of ribosomes; or the reconstituted particle is used as a purified reconstituted RNP particle which is substantially free of ribosomes.

The reconstituted particle is useful for cleaving one strand of a double-stranded DNA substrate and attaching an RNA molecule to the cleaved strand at the cleavage point; cleaving both strands of the double-stranded DNA substrate and catalyzing the insertion of an RNA molecule into the cleavage site of one strand of the DNA substrate; cleaving both strands of a double-stranded DNA substrate and attaching a nucleic acid molecule to both strands at the cleavage points. The reconstituted RNP particle preparation comprises an exogenous excised group II intron RNA, an RNA-protein complex, and a reconstituted RNP particle which comprises the exogenous group II intron RNA bound to a group II intron-encoded protein.

The RNA-protein complex contains a group II intron-encoded protein that is associated with RNA that is free of the excised, group

II RNA molecules having a sequence which encodes the group II intron- encoded protein. The group II intron-encoded protein comprises an X domain and a Zn domain and includes both unmodified and modified group II intron-encoded proteins. The X domain of the protein has a maturase activity. The Zn domain of the protein has Zn^2* finger-like motifs. More preferably, the group II intron-encoded protein further comprises a reverse transcriptase domain. The group II intron-encoded protein has a binding affinity for the RNA of the complex and for the exogenous excised group II intron RNA. The exogenous group II intron RNA of the RNP particle has at least one hybridizing region which is complementary to a recognition site on the substrate DNA. The exogenous RNA preferably has six domains . The exogenous RNA includes both unmodified and modified group II intron RNA molecules. Preferably, the exogenous RNA is an in vitro transcript or a derivative of an in vitro transcript of an unmodified or modified mtron group II intron. For example, the exogenous RNA may be derived by splicing from an m vitro transcript. In a preferred embodiment of the reconstituted particle, the exogenous RNA has the sequence encoded by the DNA sequence of SEQ.ID.NO.l and the group II mtron-encoded protein is a 62 kDa protein that is processed from a polypeptide having the sequence of SEQ. ID. NO. 2. Methods for Cleaving DNA and Inserting Nucleotides

The methods of the present invention and the reconstituted RNP particles are useful analytical tools for determining the location of a defined sequence a double-stranded DNA substrate. Moreover, the simultaneous insertion of the nucleic acid molecule mto the first strand of DNA permits tagging of the cleavage site of the first strand with a radiolabeled molecule. In addition, the automatic attachment of an RNA molecule onto one strand of the DNA substrate permits identification of the cleavage site through hybridization studies that use a probe that is complementary to the attached RNA molecule. An attached RNA molecule that is tagged with a molecule such as biotm also enables the cleaved strand to be affinity purified. These methods are also useful for rendering the substrate DNA nonfunctional or for changing the characteristics of the RNA and protein encoded by the substrate DNA.

While a wide range of temperatures are suitable for the methods herein, good results are obtained at a reaction temperature of from about 30°C to about 42°C, preferably from about 30° to about 37°C. A suitable reaction medium contains a monovalent cation such as Na^* or K^*, at a concentration from about 0 to about 300 mM; preferably from about 50 to about 200 mM KCl, and a divalent cation, preferably a magnesium or manganese ion, more preferably a magnesium ion, at a concentration that is less than 100 mM and greater than 1 mM. Preferably the divalent cation is at a concentration of about 5 to about 20 mM, more preferably about 10 to about 20 mM. The rate of attachment of the mtron RNA to the cleaved sense strand at 5 mM MgCl₂ is about 25% of the rate that is obtamed using 10-20 mM MgCl₂ The preferred pH for the medium is from about 6.0-8.5, more preferably about 7.5-8.0. Cleaving one strand of double stranded DNA

Double stranded DNA substrate havmg a recognition site is cleaved and RNA inserted at the cleavage site by reacting the substrate DNA with a nucleotide integrase. Suitable nucleotide ntegrases include : a mitochondrial RNP particle from mutant yeast stram io2^HHVR which comprises a modified, excised RNA from the group II mtron aI2 of the yeast mitochondrial C0X1 gene and an aI2-encoded protein that has a missense mutation m the HHVR motif; a mitochondrial RNP particle from mutant yeast strain ι°2^ConZ which comprises a modified, excised aI2 intron RNA and an aI2-encoded protein that lacks the most conserved motifs in the Zn domain; a mitochondrial RNP particle from mutant yeast strain l°2^c"^c/2 that comprises a modified, excised aI2 intron RNA and an aI2-encoded protein that has a missense mutation in the second Zn^*2 finger-like motif.

The most preferred reaction conditions for attachment of the intron RNA to the cleaved strand of substrate DNA are 100 mM KCl, 20 mM MgCl₂, pH 7.5, 5 mM DTT and 37°C. Cleaving both strands of double stranded DNA Both strands of a DNA substrate having a recognition site are cleaved by reacting nucleotide integrases with the DNA. Suitable nucleotide integrases include, for example: a mitochondrial RNP particle that comprises an excised aI2 RNA and aI2-encoded protein, a mitochondrial RNP particle that comprises all RNA and all intron- ■ encoded protein; a mitochondrial RNP particle from mutant yeast strain 1°2^YAHH which comprises a modified, excised aI2 RNA and an aI2-encoded protein that has the mutation YADD→YAHH in the reverse transcriptase domain of the protein; and a reconstituted RNP particle which comprises an exogenous excised aI2 RNA and an aI2-encoded protein. The most preferred reaction conditions for cleavage of both strands of the DNA substrate by the nucleotide integrase, are 100 mM KCl, 20 mM MgCl₂, pH 7.5, 5 mM DTT and 37°C. Under these conditions, the nucleotide integrase which comprises an excised all RNA and an all encoded protein cleaves and inserts the all RNA into the cleavage site of the sense strand of a double-stranded DNA substrate which contains the target sequence of SEQ. ID. NO. 17, in the sense strand. In this sequence, nucleotide bases 63-69 having the sequence 5'TTAATG, hereinafter designated as "IBS1," are complementary to nucleotide bases, hereinafter referred to as "EBS1, " in domain I of the all intron RNA. Nucleotide bases 57-62 which have the sequence 5'CAGTTA, hereinafter designated as "IBS 2," are complementary to other nucleotide bases, hereinafter referred to as "EBS2," in domain I of the all intron RNA. The RNP particles of this nucleotide integrase cleave the sense strand of this substrate between nucleotide 69 and nucleotide 70 of the target sequence, and cleave the antisense strand of this substrate 10 base pairs downstream from cleavage site of the sense strand.

Under these conditions, a nucleotide integrase which comprises an excised aI2 RNA and an aI2 encoded protein cleaves and inserts the aI2 RNA into the cleavage site of the sense strand of a double-stranded DNA substrate which contains the target sequence 5'TTTTAGTAGCTGGTCATGCTGTATTAATAATTTTCTTCTTAGTAATGCCTGCTTTAATAG GAGGTTTTGGT) , SEQ. ID. NO. 5, in the sense strand. In this sequence, nucleotide bases 31-36 having the sequence 5'TTTTCT, hereinafter designated as "IBS3," are complementary to nucleotide bases, hereinafter referred to as "EBS3," in domain I of the aI2 intron RNA. Nucleotide bases 24-30, which have the sequence 5'TTAATAA, hereinafter designated as "IBS4," are complementary to other nucleotide bases, hereinafter referred to as "EBS4, " in domain I of the aI2 intron RNA. The RNP particles of this nucleotide integrase cleave the sense strand of this substrate between nucleotide 36 and nucleotide 37 of the target sequence, and cleave the antisense strand of this substrate 10 base pairs downstream from cleavage site of the sense strand. Cleaving DNA. insertion of RNA and formation of cDNA Both strands of a DNA substrate having a recognition site are cleaved and an RNA attached to one strand of the DNA and a cDNA attached to the cleaved second strand of the DNA substrate by incubating the DNA with the nucleotide integrase in the presence of deoxynucleotides. Suitable nucleotide integrases include, for example: a mitochondrial RNP particle that comprises an excised aI2 RNA and aI2- encoded protein and a mitochondrial RNP particle that comprises all RNA and all intron-encoded protein.

Suitable conditions for the nucleotide integrase-catalyzed synthesis of a cDNA on the second strand of the DNA substrate include a reaction temperature of from about 30° to about 45°C, preferably from about 37° to about 42°C. The preferred reaction medium includes a monovalent cation such as Na* or K^*, at a range of 0-300 mM, preferably from about 50 mM to about 150 mM KCl, and a divalent cation, preferably a magnesium or manganese ion, more preferably a magnesium ion at a concentration range of from about 2 mM to about 20 mM. The preferred pH for the medium is about 7.5 to about 8.5. The most preferred conditions for synthesis of an elongated cDNA product are 100 mM KCl, 2 mM MgCl₂, 5 mM DTT, pH 8.5, and 37°C. The most preferred conditions for maximum cleavage of the second strand and the formation of a cDNA product is 100 mM KCl, a Mg²* concentration of 10 to 20 mM, 5 mM DTT and a pH of 7.5. Cleavage of single stranded DNA

Single stranded DNA substrate having a recognition site is cleaved and RNA inserted at the cleavage site by reacting the substrate DNA with a nucleotide integrase. Suitable nucleotide integrases include for example : a mitochondrial RNP particle that comprises an excised aI2 RNA and aI2-encoded protein and a mitochondrial RNP particle that comprises all RNA and all intron-encoded protein.

The most preferred reaction conditions for cleavage of the substrate and insertion of the intron RNA into the cleavage site by the nucleotide integrase, are 100 mM KCl, 20 mM MgCl₂, pH 7.5, 5 mM DTT and 37°C. Under these condition, the nucleotide integrase which comprises an aI2 intron RNA and an aI2 encoded protein cleaves and inserts an aI2 RNA into the cleavage site of a single-stranded DNA substrate that contains the IBS3 sequence 5'TTTTCT or the IBS4 sequence 5'TTAATAA. In contrast, the aI2 RNA alone is unable to cleave double stranded DNA or single stranded DNA at the 20 mM Mg²* concentration typically used in the methods of the present invention. Preparation of the Nucleotide Integrase

The nucleotide integrase is isolated from wild type or mutant yeast mitochondria, fungal mitochondria, plant mitochondria, chloroplasts, the proteotobacterium Azotobacter vinelandii , the cyanobacterium Calothrix, and Escherichia coli . The procedure for isolating the RNP particle preparation involves mechanically and/or enzymatically disrupting the cell membranes and or cell walls of the organisms. In the case of fungi and plants, the purification also involves separating the specific organelles, such as mitochondria or chloroplasts, from the other cellular components by differential centrifugation and/or flotation gradients and then lysing the organelles with a nonionic detergent, such as Nonidet P-40. The organelle and bacterium lysates are then centrifuged through a sucrose cushion to obtain the ribonucleoprotein (RNP) particle preparation. The RNP particles may be further purified by separation on a sucrose gradient, or a gel filtration column, or by other types of chromatography.

The reconstituted RNP particle preparation is prepared by combining an RNA-protein complex preparation with an exogenous excised group II intron RNA. The RNA-protein complex is preferably isolated from a yeast, fungi, or bacterium using the protocol for RNP particles described above. The RNA-protein complex comprises a group II intron- encoded protein and RNA that is free of the excised group II intron RNA having a sequence that encodes the group II intron-encoded protein.

The exogenous RNA preferably is made by in vitro transcription or by in vitro transcription and self-splicing of the group II intron. The exogenous RNA may also be made by isolation of the group II intron RNA from cells or organelles in which it is naturally present or from cells in which an altered intron has been inserted and expressed. The exogenous RNA is then added to a preparation containing the RNA-protein complex. Preferably, the exogenous group II intron RNA is first denatured. The exogenous RNA is added to the RNA-protein complex on ice.

Examples

The RNP particle preparations of the following examples 1-10, the RNA-protein complex of the example 12, and the preparations of comparative examples A-D were isolated from the mitochondria of the wild-type Saccharomyces cerevisiae yeast strain ID41-6/161 MATa adel lysl , hereinafter designated "161", and derivatives thereof. The mitochondria of the wild-type yeast strain 161 contains a COX1 gene that includes the group II intron all and the group II intron aI2.

The COX1 gene in the mutant yeast strains either lacks one of the group II introns or has a mutation in one of the group II introns. The excised group II intron RNA molecules and the group II intron encoded proteins are derived from the group II introns all and aI2 that are present in the wild-type and mutant yeast strains.

Figure 2 depicts several of the mutations/modifications that are present in the aI2-encoded proteins that are produced by the different mutant yeast strains. The intron composition of the COX1 gene in the different yeast strains is denoted by a convention in which a superscript "+" indicates the presence of the all intron or the aI2 intron, a superscript "o" indicates the absence of the all or aI2 intron, and other superscripts refer to specific alleles or mutations in the aI2 intron. Example 1

An RNP particle preparation was isolated from the mitochondria of the Saccharomyces cerevisiae wild-type yeast strain 161. The intron composition of the COX1 gene of the wild-type strain is l^*t2*^c. The RNP particle preparation contains an RNP particle that is derived from the all intron and includes an excised, all RNA bound to a protein encoded by all. The preparation also contains an RNP particle that is derived from the aI2 intron and that comprises a excised aI2 RNA molecule and an associated aI2-encoded protein. To prepare the RNP particle preparation, the yeast were inoculated into a 1 liter liquid culture medium containing 2% raffinose, 2% BactoPeptone from Difco and 1% yeast extract from Difco to an O.D.₅₉₅ of 1.6-1.7. The cell walls were digested with 40 mg of the yeast lytic enzyme from ICN, and the cells broken by mechanical disruption with glass beads. The nuclei and cell debris were pelleted from the lysate by centrifugation for 5 minutes in a Beckman GSA rotor at 5,000 rpm. The supernatant was removed and centrifuged in a Beckman GSA rotor at 13,000 rpm for 15 minutes to obtain a mitochondrial pellet. The mitochondria were layered on a flotation gradient consisting of a 44% sucrose solution layer, a 53% sucrose solution layer, and a 65% sucrose solution layer and centrifuged in a Beckman SW28 rotor at 27,000 rpm for 2 hours and 10 minutes. The mitochondria were collected from the 53%/44% interface and suspended in buffer containing 0.5M KCl, 50 mM CaCl₂, 25 mM Tris-HCl, pH 7.5, 5mM DTT and lysed by the addition of Nonidet P-40 to a final concentration of 1%. The mitochondrial lysate was then centrifuged in a Beckman 50Ti rotor at 50,000 rpm for 17 hours through a 1.85 M sucrose cushion in a buffer containing 0.5M KCl, 25 mM CaCl₂, 25 mM Tris-HCl, pH 7.5, 5mM DTT, to obtain a pellet of RNP particles that were largely free of mitochondrial proteins. The isolated RNP particles were resuspended in 10 mM Tris-HCl, pH 8.0 and 1 mM DTT and stored at

-70°C. The preparation may be repeatedly thawed and frozen before use. Example la Purified RNP particle 2.5 O.D.₂₆₀ of the RNP particles from example 1 in a volume of 150 μl were layered onto a 12 ml 5-20% linear sucrose gradient in a buffer consisting of 100 mM KCl, 2 mM MgCl₂, 50 mM Tris-HCl, pH 7.5, and 5 mM DTT. The gradient was centrifuged in an SW41 rotor at 4°C at 40,000 rpm for five hours. The gradient was fractionated into 35 fractions of approximately 325 μl. Fractions 12-20 contain the purified RNP particles which are substantially free of ribosomal RNA. The location of the RNP particles in the gradient fractions was independently verified by Northern hybridization with aI2 antisense RNA. The location of the small and large subunits of ribosomal RNA in ■ the gradient fractions was independently verified by ethidium bromide staining of the fractions on a 1% agarose gel. Example 2 RNP particle preparation from mutant yeast strain 1°2^*

The RNP particles comprise an excised aI2 RNA and an aI2-encoded protein. Yeast strain l⁰2*^c was obtained from Dr. Philip S. Perlman at the University of Texas Southwestern Medical Center and was prepared as described in Moran et al., 1995, Mobile Group II Introns of Yeast Mitochondrial DNA Are Novel Site-Specific Retroelements, Mol. Cell Biol. 15, 2838-38, which is incorporated herein by reference. The l°2^*c mutant strain was constructed as follows: (i) the aI2 intron from strain 161 was cloned as a C2aI-to-BamHI fragment into pBluescript KS^* obtained from Stratagene to yield pJVM4; (ii) pJVM4 was cleaved with Clal and Ndel to remove the 5' end of the insert; and (iii) an Λfspl-to- Ndel fragment that contains exons 1 and 2 of the mitochondria COXI gene plus the 5' end of aI2 from yeast strain C1036ΔI was inserted to yield plasmid p VM164. Yeast strain C1036ΔI, in which all is excised from the mitochondrial DNA, was prepared as described in Kennell et al. , 1993, Reverse transcriptase activity associated with maturase-encoding group II introns in yeast mitochondria. Cell 73, 133-146, which is incorporated herein by reference. pJVM164 was transformed into a [rho°] strain, and the l°2*^c allele was placed into an intact mitochondrial DNA by recombination. This last step is accomplished by mating to a nonreverting COXI mutant derived from mutant C1036 (strain 5B) , whose construction is described in Kennel et al. , 1993, and selecting for recombinant progeny that are capable of respiring and growing on glycerol-containing medium (GLY^*) and that contain the transformed COXI allele in place of the 5B allele.

The reactions and manipulations directed at cloning DNA, such as ligations, restriction enzyme digestions, bacterial transformation, DNA sequencing etc. were carried out according to standard techniques, such as those described by Sambrook et al. , Molecular cloning: a laboratory manual, 2nd ed. , Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y. Yeast mitochondrial transformations were also carried out according to standard techniques such as those described in Belcher et al. , 1994, Biolistic transformation of mitochondria in Saccharomyces cerevisiae, 101-115. Jn N.-S. Yang and P. Christou (ed.) Particle Bombardment Technology for Gene Transfer. Oxford University Press, New York. The RNP particle preparation was made from the mitochondria of mutant yeast strain 1°2*, as in Example 1. Example 3 RNP particle preparation from mutant yeast strain l^*c2⁰

Yeast strain l*^t2° is a derivative of the wild-type yeast strain 161. The yeast strain l^*c2° was obtained from Dr. Philip S. Perlman and was prepared as described in Kennell et al., 1993. Cell 73, 133-146. Yeast strain l*^t2° contains a segment of the COXI gene of S. diasta ticus, which lacks aI2, inserted into wild-type 161 mtDNA via mitochondrial transformation. The construction started with plasmid pSH2, which contains all from wild-type 161 and some flanking sequences cloned as a Hpall/EcoRI fragment in pBS^* (Stratagene, La Jolla, CA) . That plasmid was cleaved near the 3' end of all with Clal and in the downstream polylinker with BamHl, and the gap was filled with a Clal/BamHI fragment from S. diastaticus mitochondrial DNA (NRRL Y-2416) that contains the 3' end of all, E2, E3 and most of aI3, thus creating a 1^*2° form of the COXI gene. The plasmid containing the hybrid COX1- 1*2° segment was transformed into a ρ° derivative of strain MCC109 (MATα ade2-101 ura3-52 karl-1) by biolistic transformation. The resulting artificial petite was crossed to strain nl61/m5B, and gly^* recombinants containing the COXI 1*2° allele in the nl61 background were isolated. The hybrid all allele, which is spliced normally, differs from that of wild-type 161 by one nucleotide change, C to T, at position 2401, changing Thr₇₄₄ to Leu in the intron open reading frame. The RNP particle preparation was made from the mitochondria of mutant yeast strain 1*2° as in Example 1. The RNP particles comprise an excised all RNA molecule and an all encoded protein. Example 4 RNP particle preparation from mutant yeast strain 1"2^Y™ Yeast strain ι°2^YAHH was obtained from Dr. Philip S. Perlman and was made as described in Moran et al., 1995, Mol. Cell Biol. 15, 2838- 38., using a mutagenized pJVM164 plasmid. The allele was made by oligonucleotide-directed mutagenesis of pJVM164 which contains a 4.4 kb MspI/BamHI fragment extending from 217 nucleotides upstream of exon 1 through intron aI3 of the COXI allele. The mutagenesis changes the aI2 nucleotides 1473 to 1478 from GAT GAT to CAT CAT (D-491D-492 to HH) . The RNP particles comprise a mutated, excised aI2 RNA and an aI2- encoded protein that has the mutation YADD→YAHH in the reverse transcriptase domain of the protein. The RNP particle preparation was made from the mitochondria of mutant yeast strain ι°2^ΪAHH as in Example

1.

Example 5 RNP particles from the mutant yeast strain ι°2^p714T

The mutant yeast strain 1°2^P,MI was obtained from Dr. Philip S. Perlman and was constructed according to the procedure described in Kennell et al., 1993, Cell 73, 133-146, where it is named nl61/ml61- C1036Δ1. The RNP particles comprise a mutated, excised aI2 intron RNA molecule and an aI2-encoded protein that carries the missense mutation P₇₁₄T in the Zn domain. The RNP particle preparation was made from mitochondria of mutant yeast strain ι^o2^P714T as in Example 1. Example 6 RNP particle from mutant yeast strain χ°2^HHVR

The mutant yeast strain ι°2^HKVR was obtained from Dr. Philip S. Perlman and was made as described in Moran et al . , 1995, Mol. Cell Biol. 15, 2828-38, which is incorporated herein by reference, using a mutagenized pJVM164 plasmid. The allele was constructed by site- directed ^■mutagenesis of pJVM164. The aI2 intron has the following changes: positions 2208-2219 from CATCACGTAAGA to GCAGCTGCAGCT (H₇₃₆H₇₃₇V₇₃₈R₇₃₉ to AAAA) and A₂₂₂₇ A to T (N₇₄₂I) . This nucleotide integrase preparation comprises a mutated, excised aI2 intron RNA and an aI2- encoded protein that has a missense mutation in the HHVR motif. The RNP particle preparation was made from mitochondria of mutant yeast strain ι^o2^HavR. Example 7 RNP particle from mutant yeast strain ι°2^Con2n

The mutant yeast strain ι°2^*ConZn was obtained from Dr. Philip S. Perlman and was made as described in Moran et al., 1995, Mol. Cell Biol. 15, 2828-38, using a mutagenized pJVM164 plasmid. The allele was constructed by oligonucleotide-directed mutagenesis of pJVM164. The aI2 intron has the following changes:positions 2157-2165 changed from TTATTTAGT to TAATAATAA (L_7l9F₇₂₀S₇₂₁ to OchOchOch) . RNP particles comprise a mutated, excised aI2 intron RNA and an aI2-encoded protein that lacks the most conserved motifs in the Zn domain. The RNP particle preparation was made from mitochondria of mutant yeast strain ι^o2^ConZn. Example 8 RNP particle from mutant veast strain ι°2^c"c/1

The mutant yeast strain ι°2^c"c/1 was obtained from Dr. Phillip S. Perlman and was made as described in Moran et al. , 1995, using a mutagenized pJVM164 plasmid. The allele was constructed by site- directed mutagenesis of pJVM164. The aI2 intron has the following changes:positions 2172-2173 changed from TG to GC (C₇₂.A) and 2180-2182 changed from TTG to AGC (I₇₂₆C₇₂₇ to MA) . The RNP particles comprise a mutated, excised aI2 intron RNA and an aI2-encoded protein that has a missense mutation in the first Zn*²-finger-like motif. The RNP particle preparation was made from mitochondria of mutant yeast strain ι^o2^c-^c/1. Example 9 RNP particles from mutant veast strain ι°2^c'c/2 The mutant yeast strain ι°₂ ^c-^c/2 was obtained from Dr. Philip S. Perlman and was made as described in Moran et al. , 1995, using a mutagenized pJVM164 plasmid. The allele was constructed by site- directed mutagenesis of pJVM164. The aI2 intron has the following changes: position 2304-2305 changed from TG to GC (C₇₆₈A) and 2313-2314 changed from TG to GC (C₇₇₁A) . The RNP particles comprise a mutated excised aI2 intron RNA and an aI2-encoded protein that has a missense mutation in the second Zn*² finger-like motif. The RNP particle preparation was made from mitochondria of mutant yeast strain 1°2^{C"C 2}. Example 10 RNP particles from mutant yeast strain ι°2^H6

The mutant yeast strain, obtained from Dr. Philip S. Perlman, was made by transferring the mutagenized plasmid pJVM164 into the mitochondria of yeast strain GRF18 as described in Moran et al. , 1995. The allele was constructed by site directed mutagenesis of pJVM164 and has the sequence CATCATCATCATCATCAT inserted between nucleotides 2357 and 2358 of the aI2 intron. The RNP particle preparation was made from mitochondria of mutant yeast strain l°2^H6 according to the protocol described above for example 1. The RNP particles comprise a mutated, excised aI2 intron RNA and an aI2-encoded protein that has six histidines added to the C terminus of the aI2-encoded protein.

Example 11 RNP particles from Neurospora intermedia.

Mitochondria from the Varkud strain of Neurospora intermedia, which is available from the Fungal Genetics Stock Center, were prepared as described in Lambowitz A.M. 1979, Preparation and analysis of mitochondrial ribosomes. Meth. Enzymol. 5_9_, 421-433.. The conidia were disrupted with glass beads and the mitochondria and RNP particles isolated as described in Example 1. The RNP particles comprise an excised σoJ intron RNA and the protein encoded by the col intron. Example 12 Reconstituted RNP particle preparation A reconstituted RNP particle preparation was made by incubating an exogenous, excised, in vitro RNA transcript of the aI2 intron with an RNP preparation isolated from the mutant yeast strain ι°2^ΔD5, in which the aI2 intron RNA lacks a domain V and is therefore splicing defective. The mutant allele ι°2^ΔD5 was obtained from Dr. Philip S. Perlman and was constructed using the same procedure that was used to make yeast strain l*2*^υ5 that was described in Moran et al. 1995, except that the final mating was with yeast strain 1°2^*. The RNP preparation was isolated from ι°2^{Δ S} using the protocol described above in Example 1. The RNP preparation isolated from the mitochondria of ι°2^iD5 does not contain excised aI2 RNA but does contain aI2-encoded protein that is associated with other RNA molecules in the preparation.

The exogenous RNA was made by in vitro transcription of the plasmid pJVM4 which includes a fragment of the yeast mitochondrial COXI gene from the Clal site of the group II intron 1 (all) to the Bamf-T O 97/10362 PC17US96/14609

- 15 -

site of aI3 that has been inserted into the pBLUESCRIPT KS+ plasmid. As shown in Figure 3, which depicts the plasmid map of pJVM4, plasmid pJVM4 contains the following COXI sequences: Exon 2, aI2, Exon 3 and parts of all and aI3 sequence. The sequences are operably linked to a T3 RNA polymerase promoter. The Exon 2 and Exon 3 sequence are required for self-splicing of the aI2 intron RNA from the RNA transcript. pJVM4 was linearized with BstEII, which cuts at the 3' end of Exon 3 then 3 μg of the plasmid was incubated in 300 μl of 40 mM Tris-HCl at pH 8.0, 25 mM NaCl, 8 mM MgCl₂2 mM spermidine, 5 mM DTT 200 μM rNTPs, 182 U of RNasin from US Biochemical and 750 U of T3 RNA polymerase from BRL at 37°C for 2 hours to make the RNA transcripts. Following the incubation, the RNA transcripts were phenol extracted, purified on G-50 column, phenol extracted and precipitated with ethanol. The RNA transcripts were then incubated in 40 mM Tris-HCl at pH 7.5, 100 mM MgCl₂, 2 M NH.Cl at 40-45°C for 1 hour to allow self- splicing of the aI2 intron RNA molecules from the RNA transcripts and to obtain the splicing products. The splicing products, which include the excised aI2 RNA transcript, the ligated transcript which lacks the aI2 intron RNA, and the unspliced transcript, were desalted by passing through a G-50 column, then phenol extracted and ethanol precipitated to provide the exogenous RNA. The exogenous RNA was then resuspended to a final concentration of 1.0 μg/μl in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. A portion of the exogenous RNA was denatured by heating to 90°C for 2 minutes and then immediately put on ice. To prepare the reconstituted RNP particle preparation, 1 μl of the denatured exogenous RNA was added to 2 μl of the ιo2^Δds RNP preparation (0.025 O.D.₂₆₀ units) on ice for 2 minutes. The preparation was used immediately. Example 12a Purified Reconstituted Particles Reconstituted particles are purified from reconstituted preparation of Example 12 according to the method of la. Example 13 RNP Particle Preparation

An RNP particle preparation in which the group II intron RNA of the RNP particle has a "modified" EBS sequence that is complementary to a selected sequence on an intended single strand DNA substrate is prepared by two methods. First, oligonucleotide-directed mutagenesis of the group II intron DNA is performed by standard, well-known methods to change the nucleotides which encode the EBS sequences of the group II intron RNA. These sequences are in domain I of the group II intron RNA. The mutagenized intron DNA is then inserted into a vector, such as a plasmid, where it is operably linked to an RNA polymerase promoter, such as a promoter for T7 RNA polymerase or SP6 RNA polymerase or T3 RNA polymerase and an in vitro transcript of the modified group II intron RNA is made as described in example 12. The modified exogenous RNA transcript is then combined with an RNA-protein complex that has been isolated as described in example 12 to provide a modified reconstituted RNP particle preparation.

Alternatively, an RNP particle preparation in which the EBS sequences of the group II intron RNA are modified is prepared by site- directed mutagenesis of an organism, such as a yeast, as described in examples 3-9, and by isolation of the modified RNP particle preparation from the organism as described in example 1. Example 13a Purified Modified EBS Reconstituted Particles RNP particles in which the EBS sequences of the group II intron RNA have been modified are purified from the preparation of Example 13 according to the method of la. Example 14

An RNP particle preparation containing an RNP particle in which the loop region of domain IV of the group II intron RNA is modified, that is the loop region nucleotide sequence of domain IV differs from the nucleotide sequence of the aI2 RNA of Examples 1-10 is prepared by two methods. First oligonucleotide-directed mutagenesis of the aI2 intron DNA is performed by standard, well-known methods to change the nucleotide sequences which encode for the loop region of domain IV of the aI2 intron RNA. The mutagenized aI2 intron DNA is then inserted into a vector, such as a plasmid, where it is operably linked to an RNA polymerase promoter, such as a promoter for T7 RNA polymerase or SP6 RNA polymerase or T3 RNA polymerase and an in vitro transcript of the modified group II intron RNA made as described above in example 12. The exogenous RNA is then combined with an RNA-protein complex that has been isolated as described for example 12 to produce a modified reconstituted RNP particle preparation.

Alternatively, an RNP particle preparation in which the EBS sequences of the group II intron RNA are modified is prepared by site- directed mutagenesis of an organism, such as a yeast, as described in examples 4-10, and by isolation of the RNP particle preparation from the organism as described in example 1. IV is performed as in examples 4-10 of the aI2 intron DNA. Example 14a Purified Reconstituted Particles

Modified RNP particles are purified from the preparation of Example 14 according to the method of la. Comparative Examples

Comparative Example A

A ribonucleoprotein preparation was isolated from the mitochondria of the mutant yeast strain 1°2°, according to the protocol described in Example 1 to produce Comparative Example A. The mitochondrial COXI gene of mutant yeast strain 1°2° lacks the all and aI2 introns. Thus, the RNP preparation of Comparative Example A lacks excised all RNA, excised aI2 RNA, and the proteins encoded by all and aI2. Comparative Example B

An RNP preparation was isolated from mutant yeast strain ι°2^ΔDS according to the protocol described in Example 1 to produce Comparative Example B. The RNP preparation of Comparative Example B contains an aI2-encoded protein but lacks excised aI2 RNA. Comparative Example C

An RNP preparation was isolated from the mitochondria of mutagenized yeast strain 1°2" according to the protocol described in Example 1 to produce Comparative Example C. The mutant allele 1°2^X4 was obtained from Dr. Philip S. Perlman. The mutant allele was constructed by site-directed mutagenesis of pJVM16 and has the following changes in the aI2 intron: positions 2004-2012 changed from AAATTCGGT to GCAGCTGCT (K₆₆₈F₆₆₉G-₇₀ to AAA) . The RNP preparation of comparative example C contains a modified, excised aI2 RNA and an aI2-encoded protein that has a missense mutation in domain X. Comparative Example D

An RNP preparation was isolated from the mitochondria of mutagenized yeast strain ι°2^ΔZn to produce comparative Example D. The mutant allele ι°2^ΔZn was obtained from Dr. Philip S. Perlman. The mutant allele was constructed by site-directed mutagenesis of pJVM16 and has the following change in the aI2 intron: positions 2028-2036 changed from CCTCATTCA to TAATAATAA ( ₆₇₆H_S77S₆₇₈ to OchOchOch) . The RNP preparation of comparative example D contains a modified, excised aI2 RNA and an aI2-encoded protein that lacks the Zn domain.

Preparation of Substrate DNA For methods in which it is desired that the nucleotide integrase function as a tool for cleaving double-stranded DNA substrate, it is highly preferred that the DNA substrate have a recognition site that is complementary to the hybridizing region of the group II intron RNA of the nucleotide integrase. When such recognition site is not present in the intended substrate, it is preferably added to the substrate. Examples of substrates to which a recognition site has been added are described below. Of course, where the nucleotide integrase is being used to characterize DNA substrates or to determine if the DNA contains certain target sequences, a recognition site is not added to the DNA substrate. Plasmid pE2E3

Plasmid pE2E3 was made by cloning a 71 base pair insert consisting of the yeast mitochondrial COXI exons 2 and 3 into the Smal site of pBluescript KS+, obtained from Stratagene, La Jolla, CA to produce a 3032 base pair plasmid containing the target sequence 5 ' TTTTAGTAGCTGGTCATGCTGTATTAATAATTTTCTTCTTAGTAATGCCTGCT TTAATAGGAGGTTTTGGT) , SEQ. ID. NO. 5, in the sense strand. The insert was generated from the mitochondrial DNA of yeast strain 1°2° by PCR using the primers E2-GII-0 (5'TTTTAGTAGCTGGTCAGCTGTATT) , SEQ. ID. NO. 10, and E3-GII-0 (5'ACCAAAACCTCCTATTAAAGCAGGC) , SEQ. ID. NO. 11. The insert differs from wild-type 161 sequence at three positions: T to C at position 10 of exon 2, G to A at position 29 of exon 2, and T to A at position 23 of exon 3. Plasmid PE1E2

Plasmid pElE2 was made by cloning a 105 base pair insert consisting of a portion of exon 1 and all of exon 2 of the yeast mitochondrial COXI gene into the Smal site of pBluescript KS+, obtained from Stratagene to produce a 3066 base pair plasmid. The insert was generated from the mitochondrial DNA of yeast strain ml6l/ml6l C1036 delta 1 delta 2 whose construction is described in Kennell et al. , 1993, by using the primers EIJY (5'TAATCATTAGATTAGAATTAGCTGCACCTG) , SEQ.ID.NO. 8 and E2JY (5'AGAAAATCATTAATACAGC) , SEQ.ID. NO. 9. Plasmid PJVM159

Plasmid pJVM159 was constructed as described in Kennell et al . , 1993, Cell 73, 133-146. pJVM159 was obtained by cloning the 4.5 kb fragment of the yeast mitochondrial COXI gene of strain 1^* 2° from an Mspl site 217 nucleotides upstream of the start codon of exon 1 through the BamHl site in intron aI3 into pBluescript KS (+) .

Double-Stranded 142 Base-Pair DNA Substrate from pE2E3

A 142 base pair double-stranded DNA substrate containing exons 2 and 3 of the yeast mitochondrial COXI was generated from pE2E3 by PCR with the primers KS and SK. To prepare internally-labeled substrate, PCR was carried out in 25 μl of reaction medium containing 1 ng plasmid, 100 ng of each primer, 50 μCi [o.-³²P] -dTTP (3,000 Ci/mmole) from DuPont NEN, Boston, MA, 30 μM dTTP, 200 μM each of dATP, dCTP and dGTP and 2.5 units Taq DNA polymerase from Life Technologies for 25 cycles (94°C, 30 seconds; 55°C, 30 seconds; 72°C, 30 seconds) . After PCR, the internally-labeled product was ethanol precipitated and purified in a 1.5% agarose gel, extracted with phenol-chloroform-isoamyl alcohol having ratio of 25:24:1 and then ethanol precipitated. Double-Stranded 141 Base-Pair DNA Substrate from pE2E3

A similar 141 base pair internally-labeled double-stranded DNA substrate having a recognition site was synthesized from pE2E3 using primers T7 (5'GTAATACGACTCACTATAGGGC) , SEQ. ID. NO. 10, and HG3 (5'CAAAAGCTGGGTACCGGGCCCCCCC) , SEQ. ID. NO. 11. The PCR reactions were carried out as in the above paragraph. The amplified DNA substrate was digested with JVotl and Xhol from Life Technologies, Inc. to remove 3' end heterogeneity resulting from PCR. The amplified DNA substrate was then ethanol precipitated and purified in a 1.5% agarose gel containing 90 mM Tris-borate, pH 8.3, 2 mM EDTA to provide a pE2E3-generated, internally-labeled, 141 base pair,double-stranded, linear DNA substrate. 5' End-labeled DNA Substrates from pE2E3

A 142 base pair double-stranded DNA substrate that was labeled on the 5' end of the sense strand was generated from pE2E3 by PCR with end-labeled primer SK (5'CGCTCTAGAACTAGTGGATC) , SEQ.ID.NO.7, and unlabeled primer KS, both of which are complementary to a sequence in the polylinker. 0.2 μg of the primer SK was radiolabeled by using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and 150 μCi [γ-³²P] -ATP (3,000 Ci/mmole; DuPont (NEN). The PCR was carried out as in the above paragraph except in 100 μl of reaction medium with 4 ng plasmid template, 200 ng 5' end-labeled primer and 300 ng unlabeled primer. Following PCR, the labeled substrate DNAs were purified in a 1.5% agarose gel, extracted with phenol-chloroform-isoa yl alcohol (phenol-CIA; 25:24:1) and ethanol precipitated.

A 142 base pair double-stranded DNA substrate that was labeled on the 5' end of the antisense strand was generated from pE2E3 by PCR with 200 ng of the 5' end-labeled primer KS (5'TCGAGGTCG ACGGTATC) , SEQ.ID.NO.6, and unlabeled primer SK, both of which are complementary to a sequence in the polylinker. 0.2 μg of primer KS was radiolabeled by using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and 150 μCi [γ-³²P] -ATP (3,000 Ci/mmole; DuPont (NEN). The PCR was carried out as in the above paragraph. Following PCR, the labeled substrate DNAs were purified in a 1.5% agarose gel, extracted with phenol-chloroform-isoamyl alcohol (phenol-CIA; 25:24:1) and ethanol precipitated. 3' End-labeled DNA Substrates from PE2E3

To prepare 3' end-labeled substrates, 1.25 μg of unlabeled PCR product generated from pE2E3 with primers SK and KS was digested with Notl or EcoRI, and the recessed 3' ends were filled in with [α-³²P] -dCTP or[αr-³²P] -dTTP, respectively, and other unlabeled dNTPs by using Klenow DNA polymerase from Life Technologies, Inc. To ensure that only the desired 3' end was radiolabeled, the opposite end was then digested with EcoRI or BamHl . ouble-Stranded DNA Substrates from PE1E2

A 176 base pair internally-labeled double-stranded DNA substrate was synthesized from pElE2 using primers SK and KS. The PCR reactions were carried out as for the internally labeled 142 basepan double- stranded DNA substrate. The amplified DNA substrate was ethanol precipitated and purified in a 1.5% agarose gel containing 90 mM Tris-borate, pH 8.3, 2 mM EDTA to provide a pElE2-generated, double- stranded, linear DNA substrate. 5' End-Labeled Double-Stranded DNA Substrate From pE!E2 A 176 base pair double-stranded DNA substrate that was labeled on the 5' end of the sense strand was generated from pElE2 by PCR with 200 ng of the primer SK that was 5' end-labeled using phage T4 polynucleotide kinase and [γ-³ P] -ATP (3,000 Ci/mmole; DuPont NEN) . The PCR was carried out as in the above paragraph except in 100 μl of reaction medium with 4 ng plasmid template, 200 ng 5' end-labeled primer and 300 ng unlabeled primer. A 176 base pair double-stranded DNA^' substrate that was labeled on the 5' end of the antisense strand was generated from pElE2 by PCR with 200 ng of the primer KS that was 5' end-labeled using phage T4 polynucleotide kinase and .7-³²P] -ATP (3,000 Ci/mmole; DuPont NEN) . The PCR was carried out as above in 100 μl of reaction medium with 4 ng plasmid template, 200 ng 5' end-labeled primer and 300 ng unlabeled primer SK. Single-Stranded DNA Substrate from pE2E3

A 71 base-pair double-stranded DNA substrate having the recognition site was synthesized from plasmid pE2E3 by PCR with the oligonucleotides E2-GII-0 (5'TTTTAGTAGCTGGTCAGCTGTATT) , SEQ. ID. NO. 12, and E3-GII-0 (5'ACCAAAACCTCCTATTAAAGCAGGC) , SEQ. ID. NO. 13. The single stranded DNA substrate has the sequence 5 ' TTTTAGTAGCTGGTCATGCTGTATTAATAATTTTCTTCTTAGTAATGCCTGCTTTAAT AGGAGGTTTTGGT) , SEQ. ID. NO. 5, which is identical to the sense-strand of the double-stranded DNA substrate. The oligonucleotide was 3' end labeled with [α-³²P] -dTTP using terminal transferase from Life Technologies, Inc., according to the manufacturer's protocol. The labeled oligonucleotide was purified on a G-25 (Sigma) spin column, extracted with phenol-chloroform-isoamyl alcohol (phenol-CIA; 25:24:1), and ethanol precipitated. The specific activity was then adjusted to that of the double stranded DNA substrate by addition of unlabeled oligonucleotide. Single-Stranded RNA Substrate A 142 nucleotide RNA substrate was synthesized with phage T7 RNA polymerase from pE2E3 linearized with EcoRV. The RNA transcripts were synthesized in 25 μl of reaction medium containing 40 μCi [α-³²P] -UTP (3,000 Ci/mmol; DuPont NEN) , 30 μM UTP, and 200 μM ATP, CTP and GTP. The RNA substrate was precipitated and purified in a denaturing 5% polyacrylamide gel. The labeling conditions were formulated so that the 3' exon of the RNA substrate has the same specific activity as the 3' exons of the DNA substrates derived from pElE2. Double-Stranded DNA from Neurospora crassa A 180 base pair internally-labeled substrate was generated from Veurospora crassa strain 74A, which lacks the col intron. The PCR reaction was carried out in 25 μl of reaction medium containing 1 ng mtDNA from strain 74A, 100 ng of the primer 5'GAGTTAAGCGGACCTGGGGTGCAG, SEQ ID. NO. 14, 100 ng of the primer 5'ATTAAGTCTTGGGAATGCCATGTC, SEQ No. 15, 40 μCi [α-³²P] dTTP (3000 Ci/mmol; DuPont NEN) , 200 μM each of dATP, dCTP and dGTP, 2.5 U Taq DNA polymerase for 25 cycles of 94°C, 30 seconds; 55°C, 30 seconds; 72°C, 30 seconds. After PCR, the product was ethanol precipitated and gel-purified. The resulting substrate had t h e s e n s e s t r a n d s e q u e n c e o f 5'GAGTTAAGCGGACCTGGGGTGCAGTACATTGCAGATAATCAATTATACAA

TGCTATAATAACTGCACATGCGATCTTAATGATTTTCTTTATGGTTATGCCAGCATTAATAGGTGGATT

TGGTAATTTCTTGTTACCATTATTAGTAGGGGGTCCTGACATGGCATTCCCAAGACTTAAT,

Seq. Id.No. 16.

Characterization of the RNP particle. 0.3 O.D. ₂₆₀ of the RNP particle preparations of examples 1 and

5 were applied to a 1% nondenaturing agarose gel in 90 mM Tris borate, pH 8.3, 2 mM EDTA at 4°C and electrophoresed. The particles were transferred to a positively charged synthetic membrane from ICN by capillary transfer with 20 X SSC which contains 3M NaCl, 0.3 M Na-citrate at, pH 7.0. The membrane was rinsed at 4°C in reaction buffer containing 50 mM Tris, pH 8.5, 100 mM KCl, 2 mM MgCl₂, 5 mM DTT with 0.05% Nonidet P-40 and 0.2% bovine serum albumin for 15 minutes. The membrane was then incubated for 15 minutes at 37°C in 4 mL reaction buffer with 0.2% BSA 400 μCi [c.32P] dCTP (3000 Ci/mmol; DuPont NEN) , and 0.2 mM each of dATP, dGTP and dTTP. The reaction was chased with 0.2 mM dCTP for 15 minutes at 37°C. Finally, the membrane was washed 3 times for 15 minutes in 10% trichloroacetic acid, 1% sodium pyrophosphate, and exposed for autoradiography.

The lane of the gel containing RNP particles of example 5 demonstrated a single radiolabeled band. The appearance of a single band indicated that the aI2 protein of example 5, which has reverse transcriptase activity, migrates as a particle in the nondenaturing gel in which it is associated with an endogenous RNA molecule which acts as a template. The aI2 protein specifically copies an aI2 RNA sequence and does not efficiently copy nonspecific RNA sequences. Kennell et al., 1993, Reverse transcriptase activity associated with maturase- encoding group II introns in yeast mitochondria. Cell 7_3, 133-146. Thus, the presence of a single band shows that the aI2 protein is associated with a specific aI2 RNA molecule. The RNP particles of example 1 did not produce a similar radiolabeled band. It is believed that this is due to an inhibition of endogenous reverse transcriptase activity in wild-type cells.

The RNP particles were resolved on a gel, blotted onto a membrane and probed with a radiolabeled RNA which is complementary to the aI2 RNA. This experiment showed that aI2 RNA with the size of excised aI2 RNA ran on the nondenaturing gel as a single band which co- migrated with the reverse transcription signal. Thus, the aI2 protein is associated in RNP particles with excised aI2 RNA. A western blot was also prepared and showed that the aI2 protein that is present in the RNP particles of examples 1,2,4 5, 6,8, and 9 is a 62 kDa protein having reverse transcriptase function. Thus, the RNP particle which is isolated from the mitochondria of yeast strain l°2^*c comprises an excised aI2 RNA and an associated 62 kDa aI2-encoded protein.

Cleaving Both Strands of a Double-Stranded DNA Substrate and Inserting an RNA Molecule into the Cleavage Site of One Strand

Both strands of a double-stranded DNA substrate were cleaved at 37°C and in a medium containing 100 mM KCl, 20 mM MgCl₂, 5 mM DTT, at pH 7.5, unless otherwise noted.

Cleaving both strands of the pE2E3-derived DNA Substrate and Inserting an RNA Molecule

465 fmoles (1,950,000 cpm) of the uniformly-labeled, 141 base pair DNA substrate were reacted with 0.375 O.D. units of the RNP particles of Example 1 in a volume of 150 μl. At 0, 1, 3, 5, 10, 20, and 30 minutes after reaction, 20 μl portions were removed and the reaction terminated by adding 80 μl of 10 mM EDTA, 0.3 M NaOAc plus 2 μg linear acrylamide carrier, followed by extraction with phenol- chloroform-isoamyl alcohol (phenol-CIA; 25:24:1) and precipitation with ethanol. One-half of the precipitated products was treated with 0.1 μg RNase A from Sigma, St. Louis, MO, in 50 μl of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA for 15 minutes at 37°C. The products that had been treated with RNase A and the products that had not been treated with RNase A were then analyzed in a denaturing 6% polyacrylamide gel alongside a sequencing ladder generated from pE2E3 using the 5' end-labeled KS primer.

Only three bands were detected for the products that were not treated with RNAse A. These bands correspond to the 69 nucleotide 5' fragment of the antisense strand, the 72 nucleotide 3' fragment of the antisense strand and the 63 nucleotide 5' fragment of the sense strand of the DNA substrate. The missing 78 nucleotide product, corresponding to the 3' fragment of the sense strand, was detected only in those samples of DNA reaction products that were treated with RNase A prior to electrophoresis . These results indicate that the DNA substrate had been cleaved into four fragments and that an RNA molecule had been attached to the 3' fragment of the sense strand during reaction with the RNP particles.

Cleaving both strands of the pE2E3-derived DNA Substrate with RNP Particle Preparation of Examples 1. 2, 4 and 5

0.025 O.D.₂₆₀ units of the RNP particle preparations of examples

1, 2, 4, 5 and comparative examples A-D were reacted with 125 fmoles

(150,000 cpm) of the 142 base pair internally-labeled DNA substrate generated from pE2E3, for 20 minutes. To verify cleavage, the products were glyoxalated and analyzed in a 1% agarose gel.

A dark band of radiolabel of approximately 2.5 kb RNA was detected in the lanes containing the products that resulted from incubation of the DNA substrate with the RNP particles of Examples 1 and 2. A lighter band of RNA was detected in the lane containing the products that resulted from incubation of the DNA substrate with example 4, indicating that these RNP particles cleaved the sense strand of the substrate and catalyzed insertion of an RNA molecule into the cleavage site. The RNP particles of example 4 contain a mutated, excised aI2 intron RNA and an aI2-encoded protein which has a mutation in the reverse transcriptase domain. An even lighter band was detected in the lane containing products that resulted from incubation of the DNA substrate with the RNP particles of example 5, which contain a mutated, excised aI2 RNA and an aI2-encoded protein with a mutation in the Zn domain. No bands were detected in the lanes which contained the comparative examples. Thus RNP preparations which lack excised aI2 intron RNA or in which the intron-encoded protein lacks the nonconserved portion of the Zn domain, will neither cleave the DNA substrate nor attach an RNA.

Analyzing the DNA-RNA Reaction Products 625 fmoles (750,000 cpm) of the internally-labeled 142 base pair substrate DNA generated from pE2E3 were incubated with 0.125 O.D.₂₆₀ units of the RNP particles of Example 1 in 50 μl of medium containing 100 mM KCl, 20 mM MgCl₂, 50 mM Tris-HCl, pH 7.5, and 5 mM DTT at 37°C for 20 minutes. Then 40 μl of 10 mM EDTA, 0.3 M NaOAc and 2 μg linear acrylamide were added to the incubation mixture. The reaction products were extracted with phenol-CIA and precipitated with ethanol. The precipitated reaction products were divided into five portions and four of the portions were further treated with RNase A, alkali, Sl nuclease or DNase I. RNase A digestion was with 0.1 μg RNase A (Sigma, St. Louis, MO) in 50 μl of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA for 15 minutes at 37°C. S_α nuclease digestion was with 8 units S_{l t} nuclease in 50 μl of 30 mM NaOAc, pH 4.6, 50 mM NaCl, 1 mM ZnCl₂, and 1 μg single-stranded DNA (salmon sperm; Sigma) for 30 minutes at 25°C. DNase I digestion was with 7.5 units DNase I (Pharmacia, Piscataway, NJ) in 50 μl of 100 mM NaOAc, pH 4.6, 5 mM MgCl₂ for 30 mm at 37°C Alkali treatment was with 50 μl of 0.1 N NaOH at 50°C for 1 hour, followed by ethanol precipitation in the presence of 0.3 mM NaOAc at pH 5.2. The reactions products were then glyoxylated and analyzed m a 1% agarose gel containing 10 mM sodium phosphate, pH 7.0 followed by autoradiography of the dried gel.

The gel revealed two closely spaced bands of about 2.5-2.7 kb RNA in the lane containing the products that resulted from incubation of the DNA substrate with the RNP particles. These bands were absent from the lanes containing substrate alone. These bands were also absent from the lanes containing products that had been treated with nucleases or alkali. Thus the 2.5 kb RNA was attached to the DNA substrate during the reaction The finding that the RNA-DNA products withstand denaturation with glyoxal indicates a covalent linkage Cleavage of 3' and 5' end labeled DNA

0.025 O.D.₂₆₀ of the RNP particles of Example 1 were incubated for 20 minutes with 150,000 cpm of each of the 5' and 3' end-labeled DNA substrates generated from pE2E3, correspondmg to 250 fmoles of 5' end-labeled substrates and 200 fmoles of 3' end-labeled substrates. To verify cleavage, the products were denatured with glyoxal and analyzed m a 1% agarose gel.

Two closely spaced dark bands of RNA were detected only with the substrate labeled at the 3' end of the sense strand. This mdicates that both bands result from the ligation of the aI2 RNA to the 3' fragment of the sense strand. When the gels were exposed for a longer time, a lighter band was detected with the substrate that had been labeled at the 5' end of the sense strand. This indicates that a portion of the aI2 RNA is fully integrated mto the cleavage site of the sense strand during the 20 minute incubation. To confirm that the RNP particle preparation catalyzed the full integration of the RNA molecule into the cleavage s te of the sense strand of the substrate, the radiolabeled products were eluted from the gel, subjected to reverse transcπptase-PCR, and sequenced The PCR products mcluded the sequence of exon 2 of the yeast mitochondrial COXI gene followed by the sequence of the aI2 intron. Thus, the nucleotide integrase preparation catalyzed attachment of the excised aI2 RNA molecule to both fragments of the sense strand , i.e. full integration of the aI2 RNA molecule mto the sense-strand cleavage site. Identifying the RNA m the RNA-DNA Reaction Product

To confirm that the aI2 mtron RNA is attached to the cleaved strand of the DNA substrate, ol go-directed Rnase H digestions were performed. For this analysis, oligonucleotides complementary to aI2 RNA were hybridized to an RNA sequence, and the sample was subjected to RNase H digestion which digests RNA in a RNA-DNA hybrid, but not single-stranded RNA. 0.025 O.D.₂₆₀ of the products were isolated on a 1% native agarose gel and eluted. After precipitation, the products, corresponding to 0.025 O.D.₂₆₀ starting materials, were combined with 40 ng each of one or more oligonucleotides in 10 μl of 40 mM Tris, pH 7.3, 100 mM KCl and 2 mM DTT. The products were heated to 90°C for 2 minutes to denature the RNA and cooled on ice. 1 μl 100 mM MgCl₂, 0.5 μl RNasin (5 U) and 0.5 μl RNase H (1.4 U) were added, and the mixture was incubated for 30 minutes at 37°C. After phenol-CIA extraction and precipitation, the samples were glyoxalated and resolved on an agarose gel. The four oligonucleotides were found individually and in combination to result in a shift of migration of the products. In contrast, oligonucleotides which are complementary to exon 1, or to aI3 did not shift the migration of the products. Thus, the aI2 RNA is attached to the DNA substrate during the reaction.

Separate studies on a polyacrylamide gel, using an in vitro transcript of the aI2 RNA confirmed that both the 2.5 kb RNA-DNA product and the 2.7 kb RNA-DNA product represent two different forms of an aI2 RNA lariat attached to the cleaved fragment of the sense strand of the DNA substrate.

Identifying Nucleic Acid Sequences in Substrate DNA

The nucleotide integrase is useful to identify the presence of particular target sites in a double stranded substrate DNA or to cleave a double stranded substrate DNA which is known to possess the target site.

0.025 O.D.₂₆₀ units of the RNP particles of examples 1 and 2 were incubated for 20 minutes with 125 fmoles (150,000 cpm) of 3' end- labeled double-stranded DNA substrate derived from pE2E3 which contains the target sequence 5'TTTTAGTAGCTGGTCATGCTGTATTAATAATTTTC TTCTTAGTAATGCCTGCTTTAATAGGAGGTTTTGGT) , SEQ. ID. NO. 5, in the sense strand substrates. The products were extracted with CIA-phenol, ethanol-precipitated, glyoxylated and analyzed on 1% agarose gels.

A dark radiolabeled band of 2.5 kb RNA was detected in the products that resulted from the reaction between the RNP particles and the DNA that contained SEQ. ID. No. 5. A radiolabeled band was not detected in products resulting from incubation of RNP particles with a substrate that lacked the target sequence, or with a substrate lacking the sequence upstream of the putative cleavage site, ie. nucleotides 1- 35 or with a substrate which contained the aI2-E3 junction of the COXI gene. Only a faint radiolabeled band was detected when the RNP particles were incubated with a modified substrate which contains a 4 base pair substitution in IBS, or with a modified substrate that contained a 10 base pair insert at the putative cleavage site, i.e. between nucleotides 36 and 37, or with a substrate that lacked nucleotides 37-71, or with a substrate that contained the E2-aIl junction from the COXI gene.

Thus the hybridizing IBS sequences in the substrate DNA are necessary for cleavage of the sense strand of the DNA substrate by the nucleotide integrase of examples 1 and . These results also indicate that the presence of additional sequences downstream of the cleavage site in the DNA substrate will impair cleavage of the substrate by the RNP particles of examples 1 and 2. Cleaving both strands of the PE2E3-derived DNA Substrate and Inserting an RNA Molecule into the Cleavage Site of the Sense Strand Using the Purified RNP Particles

125 fmoles (150,000 cpm) of the internally-labeled 142 base pair substrate DNA generated from pE2E3 were incubated with 10 μl of each of the fractions obtained from the sucrose gradient in Example la. Taking into account the composition of the fractions, the final reaction medium of 20 μl contained 100 mM KCl, 20 mM MgCl₂, 50 mM Tris-HCl, pH 7.5, and 5 mM DTT. Following a 20 minute reaction at 37°C for 20 minutes, 30 μl of water, 5 μl 0.3 M NaOAc and 5 μg tRNA were added to the fractions. The reaction products were extracted with phenol-CIA and precipitated with ethanol, glyoxalated and analyzed on a 1% agarose gel containing 10 mM sodium phosphate, pH 7.0 followed by autoradiography of the dried gel . The radiolabeled bands at 2.5 kb were quantified with a Molecular Dynamics Phosphorimager.

Radiolabeled bands of 2.5 kb were detected when the DNA substrate was reacted with each of the fractions. Thus, the purified RNP particles of example la are used to cleave both strands of a double-stranded DNA substrate and to insert the aI2 intron RNA into the cleavage site. Cleaving Both Strands of a pE2E3 derived double-stranded DNA substrate with the Reconstituted RNP Particle Preparation and with the RNP Preparation of Example 10

The reconstituted RNP particle preparation of Example 12 was reacted with 250 fmoles (300,000 cpm) of the 142 base pair DNA substrates generated from pE2E3 and which were 5' end-labeled on either the sense strand or the antisense strand for 20 minutes at 37°C. To verify cleavage of both strands of the substrate, the reaction products were extracted with phenol-CIA in the presence of 0.3 M NaOAc and 2 μg single-stranded salmon sperm DNA followed by precipitation with ethanol. DNA reactions products were analyzed in a 6% polyacrylamide/8 M urea gel, alongside dideoxy sequencing ladders generated from pE2E3 using 5' end-labeled primers KS or SK.

Radiolabeled bands of DNA corresponding to the 5' fra ment of the sense strand and to the 5' fragment of the antisense strand were detected for the products resulting from reaction of reconstituted particle preparation and the 5' end-labeled DNA substrates. Thus the reconstituted particle preparation cleaves both strands of the DNA substrate derived from pE2E3. Similar results, i.e. cleavage of both strands, were obtained when the 5' end labeled substrates were incubated with the RNP particle preparation of example 10.

Cleaving both strands of DNA Substrate Derived from pE!E2 and Inserting an RNA Molecule into the Cleavage Site of the Sense Strand

100 fmoles (150,000 cpm) of the internally-labeled pElE2-derived substrate were incubated with 0.025 O.D.₂₆₀ of the RNP particle preparations of either Example 1 or Example 2 in a medium containing 50 mM KCl, 10 mM MgCl₂, 50 mM Tris-HCl at pH 7.5, and 5 mMDTT for 20 minutes. A portion of the DNA incubation products were digested with nucleases. The incubation products were extracted with phenol-CIA and precipitated with ethanol. The DNA incubation products were glyoxylated and applied to a 1% agarose gel containing 10 mM sodium phosphate at pH 7.0. The gel was electrophoresed and dried. The dried gel was autoradiographed.

Two radiolabeled bands of approximately 2.5 kb RNA were detected on the gel when the DNA substrate was incubated with the RNP particles of Example 1. These bands were not detected for the incubation products that were treated with RNase A or alkali. These results indicate that the excised all RNA molecule of approximately 2.5 kb, was attached to the 176 base pair DNA substrate during incubation with the RNP particles of example 1. Radiolabled bands were not detected when substrate DNA was incubated with the RNP particles of Example 2. Cleavage of End Labeled DNA Substrates

To further characterize the DNA-RNA products, 0.025 O.D.₂₆₀ of the RNP particles of example 1 were incubated with 150,000 cpm of each of the 5' and 3' end-labeled DNA substrates generated from pElE2, corresponding to 250 fmoles of 5' end-labeled substrates and 200 fmoles of 3' end-labeled substrates. Following a 20 minute incubation, the products were denatured with glyoxal and analyzed in a 1% agarose gel.

Radiolabeled bands of approximately 2.5 kb RNA were detected when the RNP particles were incubated with the 5' and 3' end-labeled DNA substrates which indicated that a complete integration of the RNA molecule into the cleavage site of the sense strand had occurred. Radiolabeled bands were not detected when the RNP particles of example 1 were incubated with substrate that had been radiolabeled on either the 5' end or the 3' end of the antisense strand of the substrate DNA. Analyzing the RNA-DNA Product formed by reaction of the PE1E2 derived DNA substrate with Nucleotide Integrase

The products that resulted from reacting an internally-labeled pElE2-derived substrate with the RNP particles of Example 1 were further analyzed on a denaturing 3.5% polyacrylamide gel (39:1 acrylamide-bisacrylamate) , using an excised aI2 RNA molecule as a marker. Three bands were detected on the autoradiograph of the gel. The location of the two, slower-migrating bands suggested that they represented a radiolabeled 3' sense-strand fragment attached to an all lariat RNA molecule, while the location of the third, faster-migrating band was consistent with the migration of a linear molecule with the length of all. Since reacting the RNP particles of example 1 with a 5'end labeled substrate derived from pElE2 also results in this band, it indicates that the reaction results in a complete integration of the all RNA into the cleavage site of the sense strand.

Next, to confirm that the RNP preparation of example 1 catalyzed the full integration of the RNA molecule into the cleavage site of the sense strand of the substrate, the radiolabeled products were eluted from the gel. The products were subjected to reverse transcriptase-PCR in which the eluted RNA was first reverse transcribed using a primer complementary to all RNA. The resulting cDNA amplified by PCR using one primer complementary to the RNA sequence and one primer complementary to the vector. The PCR products were gel purified, cloned mto pKS (+) digested with Sma 1 and sequenced according the method of Sanger. The PCR products included the sequence of exon l of the yeast mitochondrial COXI gene followed by the sequence of the all intron. The PCR products also included the sequence of the aI2 tron followed by the sequence of exon 2 of the yeast mitochondrial COXI gene. Thus, the nucleotide integrase catalyzed attachment of the RNA molecule to both fragments of the sense strand , i.e. full integration of the RNA molecule into the sense-strand cleavage site. Cleaving Both Strands of a Double-Stranded DNA Substrate and Inserting RNA nto the cleavage site of one DNA strand and a cDNA mto the Cleavage Site of the Second Strand The nucleotide integrases that cleave both strands of double stranded DNA and insert an RNA into the cleavage site of one strand are also employed m the method for inserting cDNA m the second strand The method for inserting the cDNA mto the second strand is quite similar to the method for cutting the two strands and inserting an RNA except where a cDNA is desired deoxynucleotides must be present in the reaction mixture.

Cleavage of Substrate pJVM159 and Attachment of a cDNA

0.025 O.D._26o units of the RNP particle preparations of examples 1, 2, 4 and 5 and of the materials of comparative Examples A and B were combined with 1 μg of plasmid pJVM159 m 10 μl of reaction medium. The reaction medium contamed 0.2 mM each of dATP, dGTP and dTTP, 10 μCi [< -³²P] -dCTP (3,000 Ci/mmole; DuPont NEN, Boston, MA), 100 mM KCl, and 5 mM dithiothreitol, 2 mM MgCl₂, and 50 mM Tris-HCl, pH 8.5. The reaction was initiated by addition of the RNP preparations, incubated for 10 minutes at 37°C, and chased with 0.2 mM dCTP for another 10 minutes. After the chase period, the reactions were terminated by extraction with phenol-CIA (phenol-chloroform-isoamyl alcohol; 25:24:1) in the presence of 0.3 M sodium acetate, pH 7.8, and 5 μg E. coli tRNA carrier (Sigma, St. Louis, MO) . Products were ethanol precipitated twice and resolved in 1% agarose gels containing 90 mM Tris-borate, pH 8.3, 2 mM EDTA and 0.05% ethidium bromide. The gels were dried and autoradiographed.

Two additional radiolabeled bands were detected in the lanes containing the products that resulted from incubation of the substrate DNA and the RNP particles of examples 1 and 2, as compared to the control which lacked substrate DNA. This result indicates that the RNP particles of examples 1 and 2 catalyze the formation of a DNA molecule on the cleaved DNA substrate. Such additional bands were not detected when the substrate was incubated with the material of comparative example A, with the RNP particles of Example 4, with the RNP particles of example 5 or the material of comparative example B. Thus, a nucleotide integrase which lacks an excised group II intron RNA or which contains a group II intron-encoded protein that lacks a reverse transcriptase domain does not catalyze the formation of a cDNA molecule on the cleaved strand. Cleavage of pE2E3 substrate and attachment of a cDNA

Four μg of plasmid pE2E3 were reacted with 0.100 O.D.-,-₀ units of the RNP particle preparation of example 1 in 40 μl of reaction medium containing 0.2 mM each of dATP, dGTP and dTTP, 40 μCi [α-³²P] -dCTP (3,000 Ci/mmole; DuPont NEN, Boston, MA), 100 mM KCl, 5 mM dithiothreitol, 5 mM MgCl₂, and 50 mM Tris-HCl, pH 7.5. The reaction was initiated by addition of the RNP particle preparations, incubated for 10 minutes at 37°C, and chased with 0.2 mM dCTP for another 10 minutes. The reaction products were phenol extracted, precipitated and divided into four parts. One part was incubated for 15 minutes at 37°C in 50 μl of low salt buffer containing 0.1 μg RNase A from Sigma, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. Another part was incubated for 30 minutes at 37°C in 50 μl of medium containing 2.7 units RNase H from Life Technologies, Inc., 100 mM KCl, 10 mM MgCl₂, 20 mM Tris-HCl, pH 7.5, and 0.1 mM DTT. The products were extracted with phenol-CIA (phenol-chloroform-isoamyl alcohol; 25:24:1) in the presence of 0.3 M sodium acetate at pH 7.8, and 5 μg E. coli tRNA carrier (Sigma, St. Louis, MO), ethanol precipitated twice and resolved in 1% agarose gels containing 90 mM Tris-borate, pH 8.3, 2 mM EDTA and 0.05% ethidium bromide. The gels were dried and autoradiographed.

RNase A digestion of the ³²P-labeled plasmid products carried out under low salt conditions to degrade both single stranded and double- stranded RNA, reduced most of the ³²P-labeled products to a single predominant band, which migrated close to linear pE2E3. This most likely resulted from removal of the RNA template. RNase H, which is specific for RNA-DNA duplexes, also increased the mobility of the ³²P- labeled products, which indicates that the template RNA is based paired to DNA.

Dideoxy-sequencing of the products showed that the first nucleotide incorporated was the A residue ten nucleotides downstream of the intron RNA insertion site, i.e., cDNA formation began at the antisense strand cleavage site. The sequencing ladders of the longer cDNAs showed that the 3' OH of the 5' fragment of the antisense strand is used as the primer for the formation of the cDNA and that the cDNA synthesis is initiated just downstream of the intron RNA and extends into domain VI of the intron RNA. Cleaving One Strand of Double-Stranded DNA With the RNP Particles of Examples 6. 7, 8, and 9

0.025 O.D.₂₆₀ units of the RNP particles from examples 1,2,4,5,6,7,8,9, and of the material of comparative examples A-D were incubated with 250 fmoles (300,000 cpm) of the 142 base pair DNA substrates that were generated from pE2E3 and which were 5' end-labeled on either the sense strand or the antisense strand. For comparison, the 5' end labeled DNA substrate was also incubated the RNP particle preparation of Example 1 that had been boiled prior to incubation, or had been treated with 1 μg of RNase A at 37°C for two minutes prior to incubation, or had been treated with 1 μg of protease K at 37°C for 2 minutes prior to incubation. Following a 20 minute incubation at 37°C, and the DNA incubation products were extracted with phenol-CIA in the presence of 0.3 M NaOAc and 2 μg single-stranded salmon sperm DNA followed by precipitation with ethanol. DNA incubation products were analyzed in a 6% polyacrylamide/8 M urea gel, alongside dideoxy sequencing ladders generated from pE2E3 using 5' end-labeled primers KS or SK.

A radiolabeled band corresponding to the 5' fragment was detected when RNP particles of examples 1 and 2 were incubated with substrates that had been labeled on the 5' end of either the sense strand or the antisense strand of the DNA substrate, indicating that these particles cleaved both strands of the DNA substrate. The RNP particles of Example 1 cleaved the sense strand precisely at the exon 2-exon 3 junction of the sense strand. The RNP particles of examples 1 and 2 cleaved the antisense strand 10 base pairs downstream from the sense strand cleavage site. RNP particles of Example 1 that had been treated with protease K, or RNase A, or boiled did not cleave either strand.

Radiolabeled bands were also detected when the RNP particles of Example 4 were incubated with DNA substrates that had been 5' end- O 97/10362 PO7US96/14609

- 31 -

labeled on either the sense strand or antisense strand, indicating that this nucleotide integrase cleaved both strands of DNA substrate. The RNP particles of example 4 contain a modified, excised aI2 RNA and an aI2-encoded protein which lacks detectable reverse transcriptase activity. Although the extent of cleavage of RNP particles of Example 4 is somewhat reduced compared to cleavage with the RNP particle preparation of Example 1, the endonuclease activity of the RNA is present even when the reverse transcriptase activity of the aI2-encoded protein is absent. The radiolabeled bands were detected when the RNP particles of

Example 5 were incubated with the DNA substrate that had been labeled on the 5' end of either the sense strand or antisense strand. In quantitative assays normalized by either O.D.₂₆₀ or soluble aI2 reverse transcriptase activity, the cleavage activities for the antisense and sense strands by the RNP particles of Example 5 were 6% and 25%, . respectively, of activities of the RNP particles of Example l.

A radiolabeled band corresponding to the 5' fragment was detected when the DNA substrate labeled on the 5' end of the sense strand was incubated with the RNP particles of example 6, but a band corresponding to the 5' fragment of the sense strand was not detected when the RNP particles of example 6 were incubated with a DNA substrate that had been labeled on the 5' end of the antisense strand. The RNP particles of example 6 contain a modified, excised aI2 intron RNA and an aI2-encoded protein that has an alteration in one of the putative endonuclease motifs. Similar results were obtained with the RNP particles of example 7, which contains a modified, excised aI2 intron RNA and an aI2-encoded protein in which the conserved portion of the Zn domain is absent. Likewise, RNP particles of examples 8 and 9, each of which contains a modified, excised aI2 intron RNA and an aI2-encoded protein in which there is a mutation in the Zn²*-like motif, cleaved the sense strand but not the antisense strand of the DNA substrate. For the RNP particles of examples 6, 7, 8, and 9, the level of sense-strand cleavage was proportional to the amount of RNA-DNA products detected in the agarose gels. These findings indicate that the antisense strand endonuclease activity of the aI2-encoded protein is associated with the Zn domain.

Comparative Example B, which lacks excised aI2 RNA, and Comparative Example D, which contains an aI2-encoded protein that lacks the Zn domain, did not cleave either DNA strand. A radiolabeled band corresponding to the 5' fragment was detected when the reconstituted RNP particle preparation of example 12 was incubated with substrates that had been labeled on the 5' end of either the sense strand or the antisense strand of the DNA substrate. These results establish that the reconstituted RNP particle preparation cleaves both strands of the DNA substrate.

Thus, both the catalytic RNA molecule of the nucleotide integrase and the intron-encoded protein are required for cleavage of both strands of the double stranded DNA. Certain modifications in the Zn domain and the X domain of intron-encoded protein disrupt the cleavage of the antisense strand of the nucleotide integrase. It is believed that the excised group II intron of the nucleotide integrase cleaves the first strand of the double-stranded DNA substrate and that the group II intron-encoded protein of the nucleotide integrase cleaves the second strand of the double-stranded DNA substrate. Cleaving Single-stranded DNA Substrates

The RNP particle preparation of Example 1 was incubated with the following DNA substrates: 71 base pair internally-labeled double- stranded DNA derived from pE2E3 ; the 3' end-labeled 71 nucleotide single-stranded DNA derived from pE2E3; and an 142 nucleotide in vitro transcript of aI2 RNA. The incubation was conducted in a medium either containing 100 mM KCl, 20 mM MgCl₂, 50 mM Tris-HCl, pH 7.5, and 5 mM DTT for 20 minutes at 37°C or in the same reaction medium supplemented to 100 mM MgCl₂ for 1 hour at 37°C. The DNA and RNA incubation products were extracted with phenol-CIA in the presence of 0.3 M NaOAc, pH 5.2 with 5 μg E. coli tRNA carrier from Sigma, and then ethanol precipitated and glyoxylated. The glyoxylated RNA and DNA products were then analyzed by electrophoresis in a 1% agarose gel and quantitated with a Molecular Dynamics phosphorimager.

Dark radiolabeled bands of approximately 2.5 kb and 2.7 kb RNA were detected for the products that resulted from incubation of the RNP particles with the double-stranded and single-stranded DNA substrates, in 20 mM Mg²*. In contrast, only a very faint band was detected for the products that resulted from incubation of the RNP particles with the

RNA substrate in 20 mM Mg^2*. Time course experiments showed that the relative rates of RNA insertion into the substrate by the RNP particle preparation at 20 mM Mg^2* were 32:62:1 for the double-stranded DNA, single stranded DNA and RNA, respectively. Thus, the preferred substrate for the nucleotide integrase is a double-stranded or single- stranded DNA substrate.

When the magnesium ion concentration of the medium was increased to 100 mM, only faint radiolabeled bands were detected for the products that resulted from incubation of the RNP particles with the double- stranded and single-stranded DNA substrates, which indicates that the insertion of an RNA molecule into the cleavage site of double-stranded DNA or single- stranded DNA works better at a Mg^2* concentration below 100 mM. Cleaving Substrate with IBS Recognition Site

The nucleotide integrase is useful to identify the presence of particular target sites in a single stranded substrate DNA or to cleave a single stranded substrate DNA which is known to possess the target site.

0.025 O.D.₂₆₀ units of the RNP particles of example 1 were with the 3' end-labeled single stranded DNA substrate derived from pE2E3 that contained the target sequence, 5'TTTTAGTAGCTGGTCATGCTGTAT TAATAATTTTCTTCTTAGTAATGCCTGCTTTAATAGGAGGTTTTGGT) , SEQ. ID. NO. 5 and with three single stranded DNA substrates that contained modified target sequences. Following a 20 minute incubation, the products were extracted with CIA-phenol, ethanol-precipitated, glyoxylated and analyzed on 1% agarose gels.

A dark radiolabeled band of 2.5 kb of RNA was detected the products when the nucleotide integrase and double-stranded DNA substrate derived from pE2E3 which contains the target sequence. Similarly, a radiolabeled band was detected when the RNP preparations were incubated with a single-stranded substrate that contained either IBS3, nucleotides 31 to 36 of SEQ. ID No. 5 or that contained IBS4, nucleotides 25 to 30 of SEQ. ID NO. 5. In contrast, bands were not detected when the RNP preparations were incubated with a single- stranded DNA substrate that lacked both the IBS3 and IBS4 sequence. Thus the nucleotide integrase of example 1 is useful for cleaving single stranded DNA that contains the sequence TTTTCT or TTAATAA.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Lambowitz, Alan M Zimmerly, Steven Guo, Huatao Yang, Jian

(ii) TITLE OF INVENTION: Nucleotide Integrase Preparation

(iii) NUMBER OF SEQUENCES: 17

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Calfee, Halter & Griswold

(B) STREET: 800 Superior Avenue

(C) CITY: Cleveland

(D) STATE: Ohio

(E) COUNTRY: USA

(F) ZIP: 44114

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Golrick, Mary E

(B) REGISTRATION NUMBER: 34,829

(C) REFERENCE/DOCKET NUMBER: 22727/00127

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (216) 622-8200

(B) TELEFAX: (216) 241-0816

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2483 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GTGCGCCGTT TCGCTTAATT TATCACTGTA TTGAAGTGTT AATTGATAAA CATATCTCTG 60

TTTATTCAAT TAATGAAAAC TTTACCGTAT CATTTTGGTT CTGATTATTA GTAGTAACAT 120

ACATAGTATT TAGATACGTA AACCATATGG CTTACCCAGT TGGGGCCAAC TCAACGGGGA 180 CAATAGCATG CCATAAAAGC GCTGGAGTAA AACAGCCAGC GCAAGGTAAG AACTGTCCGA 240 TGGCTAGGTT AACGAATTCC TGTAAAGAAT GTTTAGGGTT CTCATTAACT CCTTCCCACT 300 TGGGGATTGT GATTCATGCT TATGTATTGG AAGAAGAGGT ACACGAGTTA ACCAAAAATG 360 AATCATTAGC TTTAAGTAAA AGTTGACATT TGGAGGGCTG TACGAGTTCA AATGGAAAAT 420 TAAGAAATAC GGGATTGTCC GAAAGGGGAA ACCCTGGGGA TAACGGAGTC TTCATAGTAC 480 CCAAATTTAA TTTAAATAAA GCGAGATACT TTAGTACTTT ATCTAAATTA AATGCAAGGA 540 AGGAAGACAG TTTAGCGTAT TTAACAAAGA TTAATACTAC GGATTTTTCC GAGTTAAATA 600 AATTAATAGA AAATAATCAT AATAAACTTG AAACCATTAA TACTAGAATT TTAAAATTAA 660 TGTCAGATAT TAGAATGTTA TTAATTGCTT ATAATAAAAT TAAAAGTAAG AAAGGTAATA 720 TATCTAAAGG TTCTAATAAT ATTACCTTAG ATGGGATTAA TATTTCATAT TTAAATAAAT 780 TATCTAAAGA TATTAACACT AATATGTTTA AATTTTCTCC GGTTAGAAGA GTTGAAATTC 840 CTAAAACATC TGGAGGATTT AGACCTTTAA GTGTTGGAAA TCCTAGAGAA AAAATTGTAC 900 AAGAAAGTAT GAGAATAATA TTAGAAATTA TCTATAATAA TAGTTTCTCT TATTATTCTC 960

ATGGATTTAG ACCTAACTTA TCTTGTTTAA CAGCTATTAT TCAATGTAAA AATTATATGC 1020

AATACTGTAA TTGATTTATT AAAGTAGATT TAAATAAATG CTTTGATACA ATTCCACATA 1080

ATATGTTAAT TAATGTATTA AATGAGAGAA TCAAAGATAA AGGTTTCATA GACTTATTAT 1140

ATAAATTATT AAGAGCTGGA TATGTTGATA AAAATAATAA TTATCATAAT ACAACTTTAG 1200

GAATTCCTCA AGGTAGTGTT GTCAGTCCTA TTTTATGTAA TATTTTTTTA GATAAATTAG 1260

ATAAATATTT AGAAAATAAA TTTGAGAATG AATTCAATAC TGGAAATATG TCTAATAGAG 1320

GTAGAAATCC AATTTATAAT AGTTTATCAT CTAAAATTTA TAGATGTAAA TTATTATCTG 1380

AAAAATTAAA ATTGATTAGA TTAAGAGACC ATTACCAAAG AAATATGGGA TCTGATAAAA 1440

GTTTTAAAAG AGCTTATTTT GTTAGATATG CTGATGATAT TATCATTGGT GTAATGGGTT 1500

CTCATAATGA TTGTAAAAAT ATTTTAAACG ATATTAATAA CTTCTTAAAA GAAAATTTAG 1560

GTATGTCAAT TAATATAGAT AAATCCGTTA TTAAACATTC TAAAGAAGGA GTTAGTTTTT 1620

TAGGGTATGA TGTAAAAGTT ACACCTTGAG AAAAAAGACC TTATAGAATG ATTAAAAAAG 1680

GTGATAATTT TATTAGGGTT AGACATCATA CTAGTTTAGT TGTTAATGCC CCTATTAGAA 1740

GTATTGTAAT AAAATTAAAT AAACATGGCT ATTGTTCTCA TGGTATTTTA GGAAAACCCA 1800

GAGGGGTTGG AAGATTAATT CATGAAGAAA TGAAAACCAT TTTAATGCAT TACTTAGCTG 1860

TTGGTAGAGG TATTATAAAC TATTATAGAT TAGCTACCAA TTTTACCACA TTAAGAGGTA 1920

GAATTACATA CATTTTATTT TATTCATGTT GTTTAACATT AGCAAGAAAA TTTAAATTAA 1980

ATACTGTTAA GAAAGTTATT TTAAAATTCG GTAAAGTATT AGTTGATCCT CATTCAAAAG 2040

TTAGTTTTAG TATTGATGAT TTTAAAATTA GACATAAAAT AAATATAACT GATTCTAATT 2100

ATACACCTGA TGAAATTTTA GATAGATATA AATATATGTT ACCTAGATCT TTATCATTAT 2160 TTAGTGGTAT TTGTCAAATT TGTGGTTCTA AACATGATTT AGAAGTACAT CACGTAAGAA 2220

CATTAAATAA TGCTGCCAAT AAAATTAAAG ATGATTATTT ATTAGGTAGA ATGATTAAGA 2280

TAAATAGAAA ACAAATTACT ATCTGTAAAA CATGTCATTT TAAAGTTCAT CAAGGTAAAT 2340

ATAATGGTCC AGGTTTATAA TAATTATTAT ACTATTAAAT ATGCGTTAAA TGGAGAGCCG 2400

TATGATATGA AAGTATCACG TACGGTTCGG AGAGGGCTCT TTTATATGAA TGTTATTACA 2460

TTCAGATAGG TTTGCTACTC TAC 2483 (2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2448 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GTGCGCCTCT CAGTGCGTAT ATTTCGTTGA TGCGTCTAGC ATTAGTATTA TGAATCATCA 60

ATAGATACTT AAAACATATG ACTAACTCAG TAGGGGCTAA CTTTACGGGG ACAATAGCAT 120

GTCATAAAAC ACCTATGATT AGTGTAGGTG GAGTTAAGTG TTACATGGTT AGGTTAACGA 180

ACTTCTTACA AGTCTTTATC AGGATTACAA TTTCCTCTTA TCATTTGGAT ATAGTAAAAC 240

AAGTTTGATT ATTTTACGTT GAGGTAATCA GATTATGATT CATTGTTTTA GATAGCACAG 300

GCAGTGTGAA AAAGATGAAG GACCTAAATA ACACAAAAGG AAATACGAAA AGTGAGGGAT 360

CAACTGAAAG AGGAAACTCT TGAGTTGACA GAGGTATAGT AGTACCGAAT ACTCAAATAA 420

AAATGAGATT TTTAAATCAA GTTAGATACT ATTCAGTAAA TAATAATTTA AAAATAGGGA 480

AGGATACCAA TATTGAGTTA TCAAAAGATA CAAGTACTTC GGACTTGTTA GAATTTGAGA 5 0

AATTAGTAAT AGATAATATA AATGAGGAAA ATATAAATAA TAATTTATTA AGTATTATAA 600

AAAACGTAGA TATATTAATA TTAGCATATA ATAGAATTAA GAGTAAACCT GGTAATATAA 660

CTCCAGGTAC AACATTAGAA ACATTAGATG GTATAAATAT AATATATTTA AATAAATTAT 720

CAAATGAATT AGGAACAGGT AAATTCAAAT TTAAACCCAT GAGAATAGTT AATATTCCTA 780

AACCTAAAGG TGGTATAAGA CCTTTAAGTG TAGGTAATCC AAGAGATAAA ATTGTACAAG 840

AAGTTATAAG AATAATTTTA GATACAATTT TTGATAAAAA GATATCAACA CATTCACATG 900

GTTTTAGAAA GAATATAAGT TGTCAAACAG CAATTTGAGA AGTTAGAAAT ATATTTGGTG 960

GAAGTAATTG ATTTATTGAA GTAGACTTAA AAAAATGTTT TGATACAATT TCTCATGATT 1020

TAATTATTAA AGAATTAAAA AGATATATTT CAGATAAAGG TTTTATTGAT TTAGTATATA 1080

AATTATTAAG AGCTGGTTAT ATTGATGAGA AAGGAACTTA TCATAAACCT ATATTAGGTT 1140 TACCTCAAGG ATCATTAATT AGTCCTATCT TATGTAATAT TGTAATAACA TTGGTAGATA 1200

ATTGATTAGA AGATTATATT AATTTATATA ATAAAGGTAA AGTTAAAAAA CAACATCCTA 1260

CATATAAAAA ATTATCAAGA ATAATTGCAA AAGCTAAAAT ATTTTCGACA AGATTAAAAT 1320

TACATAAAGA AAGAGCTAAA GGCCCACTAT TTATTTATAA TGATCCTAAT TTCAAGAGAA 1380

TAAAATACGT TAGATATGCA GATGATATTT TAATTGGGGT ATTAGGTTCA AAAAATGATT 1440

GTAAAATAAT CAAAAGAGAT TTAAACAATT TTTTAAATTC ATTAGGTTTA ACTATAAATG 1500

AAGAAAAAAC TTTAATTACT TGTGCAACTG AACTACCAGC AAGATTTTTA GGTTATAATA 1560

TTTCAATTAC ACCTTTAAAA AGAATACCTA CAGTTACTAA AC AATTAGA GGTAAACTTA 1620

TTAGAAGTAG AAATACAACT AGACCTATTA TTAATGCACC AATTAGAGAT ATTATCAATA 1680

AATTAGCTAC TAATGGATAT TGTAAGCATA ATAAAAATGG TAGAATAGGA GTGCCTACAA 17 0

GAGTAGGTAG ATGACTATAT GAAGAACCTA GAACAATTAT TAATAATTAT AAAGCGTTAG 1800

GTAGAGGTAT CTTAAATTAT TATAAATTAG CTACTAATTA TAAAAGATTA AGAGAAAGAA 1860

TCTATTACGT ATTATATTAT TCATGTGTAT TAACTTTAGC TAGTAAATAT AGATTAAAAA 1920

CAATAAGTAA AACTATTAAA AAATTTGGTT ATAATTTAAA TATTATTGAA AATGATAAAT 1980

TAATTGCCAA TTTTCCAAGA AATACTTTTG ATAATATCAA AAAAATTGAA AATCATGGTA 2040

TATTTATATA TATATCAGAA GCTAAAGTAA CTGATCCTTT TGAATATATC GATTCAATTA 2100

AATATATATT ACCTACAGCT AAAGCTAATT TTAATAAACC TTGTAGTATT TGTAATTCAA 2160

CTATTGATGT AGAAATACAT CATGTTAAAC AATTACATAG AGGTATATTA AAAGCACTTA 2220

AAGATTATAT TCTAGGTAGA ATAATTACCA TAAACAGAAA ACAAATTCCA TTATGTAAAC 2280

AATGTCATAT TAAAACACAT AAAAATAAAT TTAAAAATAT AGGACCTGGT ATATAAAATC 2340

TATTATTAAT GATACTCAAT ATGGAAAGCC GTATGATGGG AAACTATCAC GTACGGTTTG 2400

GGAAAGGCTC TTTAACACGT GGCAACATAG GTTAATTTGC TATTTCAT 2448 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Ala Pro Thr Ser Ala Tyr Ile Ser Leu Met Arg Thr Ala Leu Val Leu 1 5 10 15

Trp Ile Ile Asn Arg Tyr Leu Lys His Met Thr Asn Ser Val Gly Ala 20 25 30 Asn Phe Thr Gly Thr Met Ala Cys His Lys Thr Pro Met Ile Ser Val 35 40 45

Gly Gly Val Lys Cys Tyr Met Val Arg Leu Thr Asn Phe Leu Gin Val 50 55 60

Phe Ile Arg Ile Thr Ile Ser Ser Tyr His Leu Asp Met Val Lys Gin 65 70 75 80

Val Trp Leu Phe Tyr Val Glu Val Ile Arg Leu Trp Phe Ile Val Leu 85 90 95

Asp Ser Thr Gly Ser Val Lys Lys Met Lys Asp Thr Asn Asn Thr Lys 100 105 110

Gly Asn Thr Lys Ser Glu Gly Ser Thr Glu Arg Gly Asn Ser Trp Val 115 120 125

Asp Arg Gly Met Val Val Pro Asn Thr Gin Met Lys Met Arg Phe Leu 130 135 140

Asn Gin Val Arg Tyr Tyr Ser Val Asn Asn Asn Leu Lys Met Gly Lys 145 150 155 ' 160

Asp Thr Asn Ile Glu Leu Ser Lys Asp Thr Ser Thr Ser Asp Leu Leu 165 170 175

Glu Phe Glu Lys Leu Val Met Asp Asn Met Asn Glu Glu Asn Met Asn 180 185 190

Asn Asn Leu Leu Ser Ile Met Lys Asn Val Asp Met Leu Met Leu Ala 195 200 205

Tyr Asn Arg Ile Lys Ser Lys Pro Gly Asn Met Thr Pro Gly Thr Thr 210 215 220

Leu Glu Thr Leu Asp Gly Met Asn Met Met Tyr Leu Asn Lys Leu Ser 225 230 235 240

Asn Glu Leu Gly Thr Gly Lys Phe Lys Phe Lys Pro Met Arg Met Val 245 250 255

Asn Ile Pro Lys Pro Lys Gly Gly Met Arg Pro Leu Ser Val Gly Asn 260 265 270

Pro Arg Asp Lys Ile Val Gin Glu Val Met Arg Met Ile Leu Asp Thr 275 280 285

Ile Phe Asp Lys Lys Met Ser Thr His Ser His Gly Phe Arg Lys Asn 290 295 300

Met Ser Cys Gin Thr Ala Ile Trp Glu Val Arg Asn Met Phe Gly Gly 305 310 315 320

Ser Asn Trp Phe Ile Glu Val Asp Leu Lys Lys Cys Phe Asp Thr Ile 325 330 335

Ser His Asp Leu Ile Ile Lys Glu Leu Lys Arg Tyr Ile Ser Asp Lys 340 345 350

Gly Phe Ile Asp Leu Val Tyr Lys Leu Leu Arg Ala Gly Tyr Ile Asp 355 360 365

Glu Lys Gly Thr Tyr His Lys Pro Met Leu Gly Leu Pro Gin Gly Ser 370 375 380 Leu Ile Ser Pro Ile Leu Cys Asn Ile Val Met Thr Leu Val Asp Asn 385 390 395 400

Trp Leu Glu Asp Tyr Ile Asn Leu Tyr Asn Lys Gly Lys Val Lys Lys 405 410 415

Gin His Pro Thr Tyr Lys Lys Leu Ser Arg Met Ile Ala Lys Ala Lys 420 425 430

Met Phe Ser Thr Arg Leu Lys Leu His Lys Glu Arg Ala Lys Gly Pro 435 440 445

Thr Phe Ile Tyr Asn Asp Pro Asn Phe Lys Arg Met Lys Tyr Val Arg 450 455 460

Tyr Ala Asp Asp Ile Leu Ile Gly Val Leu Gly Ser Lys Asn Asp Cys 465 470 475 480

Lys Met Ile Lys Arg Asp Leu Asn Asn Phe Leu Asn Ser Leu Gly Leu 485 490 495

Thr Met Asn Glu Glu Lys Thr Leu Ile Thr Cys Ala Thr Glu Thr Pro 500 505 510

Ala Arg Phe Leu Gly Tyr Asn Ile Ser Ile Thr Pro Leu Lys Arg Met 515 520 525

Pro Thr Val Thr Lys Thr Ile Arg Gly Lys Thr Ile Arg Ser Arg Asn 530 535 540

Thr Thr Arg Pro lie Ile Asn Ala Pro Ile Arg Asp Ile Ile Asn Lys 545 550 555 560

Leu Ala Thr Asn Gly Tyr Cys Lys His Asn Lys Asn Gly Arg Met Gly 565 570 575

Val Pro Thr Arg Val Gly Arg Trp Thr Tyr Glu Glu Pro Arg Thr Ile 580 585 590

Ile Asn Asn Tyr Lys Ala Leu Gly Arg Gly Ile Leu Asn Tyr Tyr Lys 595 600 605

Leu Ala Thr Asn Tyr Lys Arg Leu Arg Glu Arg lie Tyr Tyr Val Leu 610 615 620

Tyr Tyr Ser Cys Val Leu Thr Leu Ala Ser Lys Tyr Arg Leu Lys Thr 625 630 635 640

Met Ser Lys Thr Ile Lys Lys Phe Gly Tyr Aεn Leu Asn Ile Ile Glu 645 650 655

Asn Asp Lys Leu Ile Ala Asn Phe Pro Arg Asn Thr Phe Asp Asn Ile 660 665 670

Lys Lys Ile Glu Asn His Gly Met Phe Met Tyr Met Ser Glu Ala Lys 675 680 685

Val Thr Asp Pro Phe Glu Tyr Ile Asp Ser Ile Lys Tyr Met Leu Pro 690 695 700

Thr Ala Lys Ala Asn Phe Asn Lys Pro Cys Ser Ile Cys Asn Ser Thr 705 710 715 720 lie Asp Val Glu Met His His Val Lys Gin Leu His Arg Gly Met Leu 725 730 735 Lys Ala Thr Lys Asp Tyr Ile Thr Gly Arg Met Ile Thr Met Asn Arg 740 745 750

Lys Gin Ile Pro Leu Cys Lys Gin Cys His Ile Lys Thr His Lys Asn 755 760 765

Lys Phe Lys Asn Met Gly Pro Gly Met 770 775

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 785 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ala Pro Phe Arg Leu Ile Tyr His Cys Ile Glu Val Leu Ile Asp Lys 1 5 10 15

His Ile Ser Val Tyr Ser Ile Asn Glu Asn Phe Thr Val Ser Phe Trp 20 25 30

Phe Trp Leu Leu Val Val Thr Tyr Met Val Phe Arg Tyr Val Asn His 35 40 45

Met Ala Tyr Pro Val Gly Ala Asn Ser Thr Gly Thr Met Ala Cys His 50 55 60

Lys Ser Ala Gly Val Lys Gin Pro Ala Gin Gly Lys Asn Cys Pro Met 65 70 75 80

Ala Arg Leu Thr Asn Ser Cys Lys Glu Cys Leu Gly Phe Ser Leu Thr 85 90 95

Pro Ser His Leu Gly Ile Val Ile His Ala Tyr Val Leu Glu Glu Glu 100 105 110

Val His Glu Leu Thr Lys Asn Glu Ser Leu Ala Leu Ser Lys Ser Trp 115 120 125

His Leu Glu Gly Cys Thr Ser Ser Asn Gly Lys Leu Arg Asn Thr Gly 130 135 140

Leu Ser Glu Arg Gly Asn Pro Gly Asp Asn Gly Val Phe Met Val Pro 145 150 155 160

Lys Phe Asn Leu Asn Lys Ala Arg Tyr Phe Ser Thr Leu Ser Lys Leu 165 170 175

Asn Ala Arg Lys Glu Asp Ser Leu Ala Tyr Leu Thr Lys Ile Asn Thr 180 185 190

Thr Asp Phe Ser Glu Leu Asn Lys Leu Met Glu Asn Asn His Asn Lys 195 200 205

Thr Glu Thr Ile Asn Thr Arg Ile Leu Lys Leu Met Ser Asp Ile Arg 210 215 220

Met Leu Leu Ile Ala Tyr Asn Lys Ile Lys Ser Lys Lys Gly Asn Met 225 230 235 240

Ser Lys Gly Ser Asn Asn Ile Thr Leu Asp Gly Ile Asn Ile Ser Tyr 245 250 255

Leu Asn Lys Leu Ser Lys Asp Ile Asn Thr Asn Met Phe Lys Phe Ser 260 265 270

Pro Val Arg Arg Val Glu Ile Pro Lys Thr Ser Gly Gly Phe Arg Pro 275 280 285

Leu Ser Val Gly Asn Pro Arg Glu Lys Ile Val Gin Glu Ser Met Arg 290 295 300

Met Met Leu Glu Ile lie Tyr Asn Asn Ser Phe Ser Tyr Tyr Ser His 305 310 315 320

Gly Phe Arg Pro Asn Leu Ser Cys Leu Thr Ala Ile Ile Gin Cys Lys 325 330 335

Asn Tyr Met Gin Tyr Cys Asn Trp Phe Ile Lys Val Asp Leu Asn Lys 340 345 350

Cys Phe Asp Thr Ile Pro His Asn Met Leu Ile Asn Val Leu Asn Glu 355 360 365

Arg lie Lys Asp Lys Gly Phe Met Asp Leu Leu Tyr Lys Leu Leu Arg 370 375 380

Ala Gly Tyr Val Asp Lys Asn Asn Asn Tyr His Asn Thr Thr Leu Gly 385 390 395 400

Ile Pro Gin Gly Ser Val Val Ser Pro Ile Leu Cys Asn Ile Phe Leu 405 410 415

Asp Lys Leu Asp Lys Tyr Leu Glu Asn Lys Phe Glu Asn Glu Phe Asn 420 425 430

Thr Gly Asn Met Ser Asn Arg Gly Arg Asn Pro Ile Tyr Asn Ser Leu 435 440 445

Ser Ser Lys Ile Tyr Arg Cys Lys Leu Leu Ser Glu Lys Leu Lys Leu 450 455 460

Ile Arg Leu Arg Asp His Tyr Gin Arg Asn Met Gly Ser Asp Lys Ser 465 470 475 480

Phe Lys Arg Ala Tyr Phe Val Arg Tyr Ala Asp Asp Ile Ile Ile Gly 485 490 495

Val Met Gly Ser His Asn Asp Cys Lys Asn Ile Leu Asn Asp Ile Asn 500 505 510

Asn Phe Leu Lys Glu Asn Leu Ser Ile Val Met Lys Leu Asn Lys His 515 520 525

Gly Tyr Cys Ser His Gly Ile Leu Gly Lys Pro Gly Met Ser Ile Asn 530 535 540

Met Asp Lys Ser Val lie Lys His Ser Lys Glu Gly Val Ser Phe Leu 545 550 555 560

Gly Tyr Asp Val Lys Val Thr Pro Trp Glu Lys Arg Pro Tyr Arg Met 565 570 575 lie Lys Lys Gly Asp Asn Phe Ile Arg Val Arg His His Thr Ser Leu 580 585 590

Val Val Asn Ala Pro Ile Arg Arg Gly Val Gly Arg Leu Ile His Glu 595 600 605

Glu Met Lys Thr Ile Leu Met His Tyr Leu Ala Val Gly Arg Gly Ile 610 615 620

Met Asn Tyr Tyr Arg Leu Ala Thr Asn Phe Thr Thr Leu Arg Gly Arg 625 630 635 640

Ile Thr Tyr Ile Leu Phe Tyr Ser Cys Cys Leu Thr Leu Ala Arg Lys 645 650 655

Phe Lys Leu Asn Thr Val Lys Lys Val Ile Leu Lys Phe Gly Lys Val 660 665 670

Leu Val Asp Pro His Ser Lys Val Ser Phe Ser Ile Asp Asp Phe Lys 675 680 685

Ile Arg His Lys Met Asn Met Thr Asp Ser Asn Tyr Thr Pro Asp Glu 690 695 700

Ile Leu Asp Arg Tyr Lys Tyr Met Leu Pro Arg Ser Leu Ser Leu Phe 705 710 715 720

Ser Gly Ile Cys Gin Ile Cys Gly Ser Lys His Asp Leu Glu Val His 725 730 735

His Val Arg Thr Leu Asn Asn Ala Ala Asn Lys Ile Lys Asp Asp Tyr 740 745 750

Leu Leu Gly Arg Met Ile Lys Met Asn Arg Lys Gin Ile Thr Ile Cys 755 760 765

Lys Thr Cys His Phe Lys Val His Gin Gly Lys Tyr Asn Gly Pro Gly 770 775 780

Leu 785

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 71 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : TTTTAGTAGC TGGTCATGCT GTATTAATAA TTTTCTTCTT AGTAATGCCT GCTTTAATAG 60 GAGGTTTTGG T 71

(2) INFORMATION FOR SEQ ID NO:6 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 : TCGAGGTCGA CGGTATC 17

(2) INFORMATION FOR SEQ ID NO: 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : CGCTCTAGAA CTAGTGGATC 20

(2) INFORMATION FOR SEQ ID NO:8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TAATCATTAG ATTAGAATTA GCTGCACCTG 30

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AGAAAATCAT TAATACAGC 19

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CTAATACGAC TCACTATAGG GC 22

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CAAAAGCTGG GTACCGGGCC CCCCC 25

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TTTTAGTAGC TGGTCAGCTG TATT 24

(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ACCAAAACCT CCTATTAAAG CAGGC 25

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GAGTTAAGCG GACCTGGGGT GCAG 24

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: ATTAAGTCTT GGGAATGCCA TGTC 24

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 180 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

GAGTTAAGCG GACCTGGGGT GCAGTACATT GCAGATAATC AATTATACAA TGCTATAATA 60

ACTGCACATG CGATCTTAAT GATTTTCTTT ATGGTTATGC CAGCATTAAT AGGTGGATTT 120

GGTAATTTCT TGTTACCATT ATTAGTAGGG GGTCCTGACA TGGCATTCCC AAGACTTAAT 180

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 105 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TAATCATTAG ATTAGAATTA GCTGCACCTG GTTCACAATA TTTACATGGT AATTCACAGT 60 TATTTAATGT TTTAGTAGTT GGTCATGCTG TATTAATGAT TTTCT 105

Claims

What is Claimed is:

1. A method for cleaving a double-stranded or single-stranded DNA substrate having a recognition site, comprising the following steps:

(a) providing a nucleotide integrase comprising:

(i) an excised group II intron RNA having a hybridizing region with a sequence complementary to the recognition site on at least one strand of the DNA substrate; and

(ii) a protein bound to said RNA and having an amino acid sequence encoded by a group II intron;

(b) reacting said nucleotide integrase with the DNA substrate in the presence of a sufficient concentration of divalent cation so that at least one strand of the DNA substrate is cleaved and a nucleic acid molecule is attached to one or both of the cleaved strands.

2. The method of claim 1 wherein: the DNA is double-stranded; said nucleotide integrase is an RNP particle; the group II RNA further comprises six domains; only one strand of the DNA substrate is cleaved; and a nucleic acid molecule is an RNA molecule which is attached to the cleaved strand.

3. The method of claim 1 wherein the DNA is double-stranded; said nucleotide integrase is an RNP particle; the protein comprises: an X domain; and a Zn domain; each strand of the DNA substrate is cleaved; and a nucleic acid molecule is attached to at least one of the cleaved strands.

4. The method of claim 3 wherein the group II RNA further comprises six domains.

5. The method of claim 3 wherein a nucleic acid molecule attached to the cleaved DNA strand is a group II intron RNA.

6. The method of claim 3 further comprising the step of : reacting deoxynucleotides with the DNA substrate in the presence of said nucleotide integrase; wherein said protein further comprises a reverse transcriptase domain, a nucleic acid molecule is attached to each of the cleaved strands.

7. The method of claim 6 wherein a group II intron RNA is attached to one strand and a cDNA is attached to the other strand of the DNA substrate.

8. The method of claim 1 wherein: the nucleotide integrase is an RNP particle; the group II intron RNA is a yeast mitochondrial group II intron RNA and the protein amino acid sequence is encoded by a yeast mitochondrial group II intron.

9. The method of claim 1 wherein: the nucleotide integrase is an RNP particle; the RNA is a fungal mitochondrial group II intron RNA; and the amino acid sequence is encoded by a fungal mitochondrial group II intron.

10. The method of claim 1 wherein the divalent cation is a magnesium ion at a concentration from about 2 mM to about 20 mM.

11. The method of claim 1 wherein the divalent cation is a magnesium ion at a concentration less than about 100 mM.

12. The method of claim 1 wherein the divalent cation is a magnesium ion at a concentration greater than about 1 mM.

13. The method of claim 1 wherein: said nucleotide integrase is an RNP particle; the RNA is an excised RNA transcript encoded by the group II intron aI2 of the yeast mitochondrial COX2 gene; and the protein is encoded by the group II intron aI2 of the yeast mitochondrial COXI gene.

14. The method of claim 1 wherein: said nucleotide integrase is an RNP particle; the RNA is an excised RNA transcript encoded by the group II intron all of the yeast mitochondrial COXI gene; and the protein is encoded by the group II intron of the all of the yeast mitochondrial COXI gene.

15. The method of claim 1 wherein: said nucleotide integrase is an RNP particle; the RNA is an excised RNA transcript encoded by the fungal mitochondrial group II intron col; and the protein is encoded by the fungal mitochondrial group II intron col.

16. The method of claim 13 wherein the aI2 intron has the nucleotide sequence of SEQ. ID. NO. 1; said RNA transcript is about 2.5 kb; and said protein has a molecular weight of about 62 kDa.

17. The method of claim 14 wherein the all intron has the nucleotide sequence of SEQ. ID. NO. 3.

18. The method of claim 1 wherein the DNA is double-stranded and the nucleotide integrase is provided in a reconstituted RNP particle preparation which comprises:

(i) an exogenous RNA which comprises the excised group II intron RNA having a hybridizing region having a sequence complementary to the recognition site on at least one strand of the DNA substrate; and (ii) an RNA-protein complex comprising: the protein having an amino acid sequence encoded by group II intron further comprising, an X domain and a Zn Domain; and RNA which is free of excised group II intron RNA that encodes said protein; and (iii) a reconstituted RNP particle comprising said exogenous RNA bound to said protein.

19. The method of claim 18 wherein said exogenous RNA comprises an excised RNA encoded by the group II intron aI2 of the yeast mitochondrial COXI gene; and said protein is encoded by the group II intron aI2 of the yeast mitochondrial COXI gene.

20. The method of claim 1 wherein the DNA is single-stranded and the excised group II intron RNA has a hybridizing region complementary to at least one recognition site on the single-stranded DNA substrate.

21. A reconstituted RNP particle preparation, comprising:

(a) an exogenous RNA which comprises an excised group II intron RNA; and

(b) an RNA-protein complex comprising:

(i) a protein having an amino acid sequence encoded by a group II intron, an X domain and a Zn domain, and

(ii) RNA that is free of excised group II intron RNA having a sequence that encodes said protein; and

(c) a reconstituted RNP particle that cleaves DNA at a specific site, said RNP particle comprising said exogenous RNA bound to said protein.

22. The reconstituted RNP particle preparation of claim 21 wherein: the exogenous RNA comprises a yeast mitochondrial group II intron RNA and the protein comprises an amino acid sequence encoded by a yeast mitochondrial group II intron.

23. The reconstituted RNP particle preparation of claim 21 wherein: the exogenous RNA comprises an excised RNA transcript that is encoded by the group II intron aI2 of the yeast mitochondrial COXI gene and said protein comprises an amino acid sequence encoded by the group

II intron aI2 of the yeast mitochondrial COXI gene.

24. A purified reconstituted RNP particle comprising:

(a) an exogenous RNA which comprises an excised group II intron RNA; and

(b) a protein having an amino acid sequence encoded by a group II intron bound to said RNA; wherein said RNP particle is substantially free of ribosomes.

25. A method for making the reconstituted RNP particle preparation of claim 21 comprising the following steps:

(a) providing an exogenous RNA molecule which comprises an excised group II intron RNA;

(b) providing an RNA-protein complex comprising:

(c) combining said exogenous RNA with said RNA-protein complex to form a reconstituted RNP particle.

26. The method of claim 25 wherein the exogenous RNA comprises a yeast mitochondrial group II intron RNA and the protein comprises an amino acid sequence encoded by a yeast mitochondrial group II intron.

27. The method of claim 25 wherein the exogenous RNA comprises an excised RNA transcript that is encoded by the group II intron aI2 of the yeast mitochondrial COXI gene and said protein comprises an amino acid sequence encoded by the group II intron aI2 of the yeast mitochondrial COXI gene.

28. A purified RNP particle, comprising: (i) an excised group II intron RNA having a hybridizing region having a sequence which is complementary to a recognition site on one strand of a DNA substrate;

(ii) a protein bound to said group II intron RNA, said protein comprising an amino acid sequence which is encoded by a group II intron, an X domain and a Zn domain; and wherein said RNP particle is substantially free of ribosomal RNA.

29. The purified RNP particle of claim 28 wherein said group II intron RNA has six domains and said protein further comprises a reverse transcriptase domain.

30. The purified RNP particle of claim 29 wherein said RNA comprises a yeast mitochondrial group II intron RNA and said protein comprises an amino acid sequence encoded by a yeast mitochondrial group II intron.

31. The method of claim 1 wherein the nucleotide integrase is reacted with the DNA substrate in the presence of from about 50 mM to about 300 mM KCl and from about 2 mM to about 20 mM Mg²*.