JP2016077180A - RecA RECOMBINANT ENZYME AND STRAIGHT CHAIN DUPLEX DNA POLYMER FORMATION TECHNIQUE USING PROTEIN WITH RECOMBINANT ACTIVITY - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing polymeric straight chain duplex DNA by promoting the binding between DNA molecules without circularizing a plurality of straight chain duplex DNAs within the same molecule.SOLUTION: The invention discloses a method for producing duplex DNA in which a plurality of straight chain duplex DNAs are linked in series by incubating the plurality of straight chain duplex DNAs with DNA ligase under the presence of RecA family recombinant enzyme or a protein with recombinant activity to inhibit the binding of both ends within the straight chain duplex DNA molecule, and to promote the binding between the ends of straight chain duplex DNA molecules.SELECTED DRAWING: None

Description

  The present invention relates to a method for suppressing the circularization in the same DNA molecule and promoting the linkage between DNA molecules when linking a plurality of linear double-stranded DNAs in series.

  The technology in the field of genetic engineering is making remarkable progress every day, and provides indispensable tools in the development of new medicines and foods. One of the basic steps of genetic manipulation is to prepare a DNA having a desired sequence. In particular, in order to construct a DNA having a desired sequence by linking a plurality of different linear double-stranded DNA molecules, the DNA molecules need to be bound in a desired order. One of the enzymes used in this step is DNA ligase. DNA ligase is an enzyme that connects the ends of DNA strands with a phosphodiester bond, but does not necessarily connect ends between different DNA molecules but also connects ends within the same DNA molecule. Therefore, when DNA ligase is used to connect a plurality of different DNA molecules, circular DNA is also generated due to the bonding of the ends of the same molecule, in addition to the linear DNA generated by the connection between the DNA molecules. Such circularization of DNA is one of the factors that reduce the production efficiency of desired linear DNA.

  So far, many studies have been conducted to suppress the circularization in the same DNA molecule that occurs when linking linear double-stranded DNA. For example, in the presence of a polymer compound that performs molecular crowding, it has been reported to promote the ligation of DNA molecules at the blunt end and sticky end by DNA ligase (Non-Patent Document 1 and Non-Patent Document). 2). However, under the conditions using molecular crowding as described above, binding between the 3 'end and the 5' end in the same molecule is also promoted, and it has been difficult to suppress the phenomenon of DNA circularization. In addition, Hayashi et al. Reported that when a high concentration of monovalent cation was added in the presence of polyethylene glycol 6000 (PEG6000), DNA ligase promoted binding between DNA molecules, resulting in linear DNA multimers ( Non-patent document 3). However, according to the method of Hayashi et al., Although it was possible to prevent circularization of DNA monomers, it was not clarified whether circularization of multimerized DNA could be prevented. Furthermore, Rusche et al. Reported that the binding reaction between linear double-stranded DNAs by T4 DNA ligase is promoted in the presence of RecA (Non-patent Document 4). However, since T4 DNA ligase itself is ATP-requiring under the conditions in which RecA and T4 DNA ligase are reacted, it is unclear as to whether ATP is required in the ligation reaction of double-stranded DNA with RecA. In addition, under the conditions disclosed in Non-Patent Document 4, at most, a trimeric linear double-stranded DNA is produced, and it is difficult to prepare a tetrameric or higher linear double-stranded DNA. is there.

  As described above, further research and development is necessary to efficiently produce multimeric linear double-stranded DNA by preventing the circularization of multiple linear double-stranded DNA within the DNA molecule. Was in the situation.

Pheiffer et al., Nucleic Acids Res., 11: 7853-7871 1983. Zimmerman et al., Proc. Natl. Acad. Sci. USA., 80: 5852-5856 1983. Hayashi et al., Nucleic Acids Res., 13: 3261-3271 1985. Rusche et al., J. Biol. Chem., 260: 949-955 1985.

  It is an object of the present invention to produce a multimeric linear double-stranded DNA by preventing a plurality of linear double-stranded DNAs from circularizing the same molecule and promoting the binding between DNA molecules.

  In the presence of RecA family recombination enzymes RecA, Rad51, and Rad52, a protein having recombination activity, when the linear double-stranded DNA is ligated by DNA ligase, the linear double-stranded DNA The present inventors have completed the present invention by finding a condition that the binding between the DNA molecules is promoted and the circularization of the DNA molecules due to the bonding of the ends in the linear double-stranded DNA molecule is suppressed.

That is, this invention is the following (1)-(12).
(1) Incubating multiple linear double-stranded DNAs with DNA ligase in the presence of a RecA family recombinase or a protein having recombination activity, thereby binding the ends of the linear double-stranded DNA molecules to each other. A method for producing a double-stranded DNA in which a plurality of linear double-stranded DNAs are connected in series by promoting and suppressing binding at both ends in the linear double-stranded DNA molecule.
(2) The method according to (1) above, wherein the end to which the linear double-stranded DNA molecule is bonded is a sticky end.
(3) The method according to (1) above, wherein the end to which the linear double-stranded DNA molecule is bonded is a blunt end.
(4) The RecA family recombinase is selected from the group consisting of RecA and Rad51, and the protein having recombination activity is Rad52, according to any one of (1) to (3) above Method.
(5) The method according to (4) above, wherein the RecA family recombinase is RecA.
(6) The method according to (5) above, which comprises incubating in the absence of ATP.
(7) The method according to (5) above, further comprising incubation under conditions to which ATP and a molecule having a molecular crowding effect are added.
(8) The method according to (1) above, wherein the RecA family recombination enzyme is Rad 51 and incubated in the presence of ATP.
(9) The method according to (1) above, wherein the protein having recombination activity is Rad52.
(10) The method according to (8) or (9), further comprising incubating under conditions to which a molecule having a molecular crowding effect is added.
(11) The method according to (7) or (10) above, wherein the molecule having a molecular crowding effect is polyethylene glycol.
(12) A kit comprising a RecA family recombinase or a protein having recombination activity, or an expression vector of a RecA family recombinase or a protein having recombination activity, comprising a plurality of linear duplexes A kit for preparing double-stranded DNA in which DNA is linked in series.

  According to the present invention, circularization of linear double-stranded DNA can be suppressed and a plurality of linear double-stranded DNAs can be linked. As a result, a double-stranded DNA having a desired base sequence can be efficiently produced.

Results of examination of linear double-stranded DNA binding reaction in the presence of PEG (consideration of conventional technology). PUC119 double-stranded DNA with 3 'protruding single-stranded parts of 4 bases complementary at both ends, cleaved with PstI, was reacted with E. coli DNA ligase (Takara, 4U / 20 μL) at 37 ° C for 40 minutes. . As a result of performing the reaction in the presence or absence of ATP, adding the indicated concentration of PEG6000, and subjecting the reaction product to agarose gel electrophoresis without Etidium bromide (A) or in the presence of 0.06 μM Etidium bromide (B). is there. In the figure, C indicates circular DNA, and L indicates linear DNA. Examination of the influence of monovalent cations on the binding reaction of linear double-stranded DNA in the presence of PEG (examination of conventional technology). PUC119 double-stranded DNA (6 μM) cleaved with Pst1 was added with the indicated concentrations of NaCl and 3.8, 7.5, 11.3% PEG6000, and reacted with T4 DNA ligase (20 U / μL) at 37 ° C. for 40 minutes. The reaction product is the result of agarose gel electrophoresis in the absence of Etidium bromide (A) or in the presence of 0.06 μM Etidium bromide (B). In the figure, C indicates circular DNA, and L indicates linear DNA. The DNA concentration (6 μM) refers to the concentration of nucleotides constituting DNA (the same applies hereinafter). Promoting effect of RecA on ligation reaction of linear double-stranded DNA (in the absence of PEG6000). PUC119 linear double-stranded DNA (6 μM) cleaved with PstI was reacted with NAD-dependent E. coli DNA ligase (6U / 20 μL) at 37 ° C. for 40 minutes. Thereafter, the reaction product was subjected to agarose gel electrophoresis in the presence of 0.06 μM Etidium bromide. In the figure, C indicates circular DNA, and L indicates linear DNA. Promoting effect of RecA on ligation reaction of linear double-stranded DNA (in the presence of PEG6000). In the presence of the indicated amount of PEG6000, 2 μM RecA in a reaction (37 ° C, 40 minutes) in which pUC119 linear double-stranded DNA cleaved with HindIII or PstI was ligated with NAD-dependent E. coli DNA ligase. Was added. After the reaction, the reaction product was electrophoresed on an agarose gel in the presence of 0.06 μM Etidium bromide. In the figure, L indicates linear DNA. It is the result of investigating the influence of Mg ²⁺ on the ligation reaction of linear double-stranded DNA in the presence of RecA. Ligating pUC119 linear double-stranded DNA (6 μM) cleaved with PstI in the presence of the indicated concentrations of MgCl ₂ and NAD-dependent E. coli DNA ligase and 3.75% (W / V) of PEG6000 2 μM RecA or 2 μM RecA and 1.3 mM ATP were added to the reaction (37 ° C. 40 min). After the reaction, the reaction product was electrophoresed on an agarose gel in the presence of 0.06 μM Etidium bromide. The incubation time in the ligation reaction of linear double-stranded DNA in the presence of RecA was examined. For ligation of linear double-stranded DNA with NAD-dependent E. coli DNA ligase, add PEG only (upper figure), add PEG and RecA (middle figure), and add PEG, RecA and ATP (in the middle figure) The reaction product was incubated at 37 ° C. for the indicated reaction time in the state shown below, and the reaction product was subjected to agarose gel electrophoresis in the presence of 0.06 μM Etidium bromide. It is the result of investigating a ligation reaction when the concentration of linear double-stranded DNA and the concentration of RecA were changed in the presence of PEG6000 and ATP, with a reaction time of 40 minutes. At each indicated DNA concentration, the RecA concentration was varied as indicated in the figure to perform the reaction. The reaction conditions are listed below the figure. Examination of the influence of Rad51 on the ligation reaction of linear double-stranded DNA. PUC119 linear double-stranded DNA (6 μM) cleaved with PstI was ligated with ATP-dependent T4 DNA ligase (37 ° C, 40 min) in the presence of 1.3 mM ATP and 7 mM MgCl ₂ The indicated concentration of Rad51 was added in the presence or absence of 3.75% PEG. The reaction product was subjected to agarose gel electrophoresis in the presence of 0.06 μM Etidium bromide. The upper figure is the result of agarose gel electrophoresis, the middle figure is the result of quantifying the concentration of the linear double-stranded DNA (polymer) band, and the lower figure is the concentration of the circular double-stranded DNA (supercoil) band. It is the result of quantification. The quantification of the bands was expressed with the total amount of bands (polymer, linear, supercoil) in each lane as 100%. Examination of the effect of PEG6000 on the ligation reaction of linear double-stranded DNA in the presence of Rad51. PUC119 linear double-stranded DNA (6 μM) cleaved with PstI is ligated with NAD-dependent E. coli DNA ligase (37 ° C, 40 min) in the presence or absence of 1.3 mM ATP, 7 mM MgCl ₂ present Below, 0.5 μM Rad51 was added in the presence of the indicated concentration of PEG6000. After the reaction, the reaction product was subjected to agarose gel electrophoresis in the presence of 0.06 μM Etidium bromide. Examination of the ligation promotion reaction of linear double-stranded DNA by Rad52. PUC119 linear double-stranded DNA (6 μM) cleaved with PstI is added to ATP-dependent T4 DNA ligase (in this case, ATP is added to 1.3 mM) or NAD-dependent E. coli DNA ligase (in this case) In the presence of 7 mM MgCl 2, the indicated concentration of Rad52 was added to the reaction (37 ° C., 40 minutes) ligated by adding NAD to 0.1 mM. After the reaction, the reaction product was subjected to agarose gel electrophoresis in the presence of 0.06 μM Etidium bromide. The upper figure is the result of agarose gel electrophoresis, the middle figure is the result of quantifying the concentration of the linear double-stranded DNA (polymer) band, and the lower figure is the concentration of the circular double-stranded DNA (supercoil) band. It is the result of quantification. The quantification of the bands was expressed with the total amount of bands (polymer, linear, supercoil) in each lane as 100%.

One embodiment of the present invention is to incubate a plurality of linear double-stranded DNAs with a DNA ligase in the presence of a RecA family recombinase or a protein having recombination activity. Double-stranded DNA in which a plurality of linear double-stranded DNAs are connected in series by promoting the bonding between the ends of DNA molecules and suppressing the binding of both ends in the linear double-stranded DNA molecule It is a method of producing.
Here, the RecA family recombinase is an enzyme that catalyzes a homologous DNA pairing reaction, and examples thereof include RecA protein, Rad51 protein, Dmc1 protein, and RadA protein. A protein having recombination activity is a protein known to function in the process of homologous recombination. For example, Rad51 protein paralog (Rad51B protein, Rad51C protein, Rad51D protein, Rad51L protein, Rad51L2 protein, Rad51D protein, Rad51L3 protein, Xrcc2 protein or Xrcc3 protein), and Rad52 protein.

As the RecA protein used in the present invention, for example, various RecA proteins described in Karlin et al., “Bacterial classifications derived from RecA protein sequence comparisons,” J. Bacteriol., 177,6881-6893 (1995) are used. Alternatively, the amino acid sequence or cDNA sequence registered in the NCBI database, for example, RecA protein and cDNA of Staphylococcus aureus represented by accession number WP_000755731, GI number 446678385, or accession number BAA04215 (amino acid In addition to the RecA protein and cDNA of Thermus thermophiles HB8 represented by D17392 (base sequence), the RecA protein represented by SEQ ID NO: 1 (SEQ ID NO: 2 is the base sequence encoding this). May be used.
The Rad51 protein and Rad52 protein of the present invention are amino acid sequences or cDNA sequences registered in NCBI (National Center for Biotechnology Information) database, for example, Rad51 of Saccharomyces cerevisiae represented by accession number CAA45563, GI number 4275. In addition to protein and cDNA, the Rad51 protein represented by SEQ ID NO: 3 used in this Example (SEQ ID NO: 4 represents the base sequence encoding this), and the Rad52 protein represented by SEQ ID NO: 5 (SEQ ID NO: 6 represents (A base sequence encoding this) may be used.

Furthermore, the RecA protein, Rad51 protein, and Rad52 protein of the present invention also include proteins substantially identical to these proteins.
For example, when RecA protein, Rad51 protein, and Rad52 protein are represented by SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, these RecA protein, Rad51 protein, and Rad52 protein are substantially the same. The identical protein is about 60% or more, preferably about 70% or more, more preferably about 80% or 81%, respectively, with the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, Most preferably a protein having an amino acid sequence having amino acid identity of about 99% and having an activity of promoting terminal binding between linear double-stranded DNA molecules by DNA ligase.

Alternatively, when the RecA protein, the Rad51 protein, and the Rad52 protein are represented by SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, the RecA protein, the Rad51 protein, and the Rad52 protein are substantially the same. In the amino acids represented by SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, one or several proteins (preferably about 1 to 30, more preferably 1 to 10), respectively. A protein having an amino acid sequence in which amino acids are deleted, substituted or added, and having an activity of promoting terminal binding between linear double-stranded DNA molecules by DNA ligase That is.
Amino acid deletions, additions, and substitutions may be mutations that originally existed in nucleic acids encoding proteins, or were newly introduced by modifying the nucleic acids by methods known in the art. May be. In the modification, for example, substitution of a specific amino acid residue can also be performed using a commercially available kit (for example, MutanTM-G (TaKaRa), MutanTM-K (TaKaRa)).
In the present specification, the terms “RecA”, “Rad51”, and “Rad52” refer to the RecA protein, the Rad51 protein, and the Rad52 protein, respectively, unless otherwise specified.

  The present invention promotes the binding between linear double-stranded DNA molecules by DNA ligase to connect linear double-stranded DNAs in series, and linear double-stranded DNA (monomer linear double-stranded DNA). The present invention relates to a method for preventing circularization of heavy chain DNA (including linear double-stranded DNA connected in series). The shape of the end of the linear double-stranded DNA used in the present invention may be either a blunt end or a sticky end, but it is preferable to connect the blunt ends or the sticky ends.

  The DNA ligase used in the present invention is an enzyme that connects the ends of DNA strands with a phosphodiester bond, and any enzyme having such an activity can be used. Is possible. Examples of DNA ligase include, but are not limited to, DNA ligase from E. coli, T4 DNA ligase from T4 phage, Pfu DNA from Pyrococcus furiosus, a hyperthermophilic marine archaea Examples include ligase, T7 DNA Ligase derived from T7 phage, DNA ligase derived from Thermus thermophiles HB8 (accession number: AAA27487 (amino acid sequence), M36417 (base sequence)).

In the present invention, the reaction conditions for linking linear double-stranded DNAs in series may be any conditions as long as they are optimally used in the enzymatic reaction of DNA ligase and are generally used. For example, a divalent cation such as MgCl ₂ , a salt such as NaCl, a reducing agent such as DTT, and a coenzyme such as ATP or NAD may be added to the solution. In particular, by appropriately adding a molecule having a molecular crowding effect, it is possible to efficiently promote the linkage of linear double-stranded DNA and prevent the circular double-stranded DNA from being circularized.
The molecule having a molecular crowding effect used in the present invention is not particularly limited. For example, polyethylene glycol (for example, PEG200, PEG400, PEG6000, PEG8000, PEG20000, etc. is not particularly limited), Ficoll, polyvinyl alcohol, polyvinyl Pyrrolidone, polyvinyl methyl ether, polyvinyl methyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, Polyaspartamide, synthetic polyamino acid, albumin, hemoglobin, sucrose, dextran, epichrome Hydrin, Fekoru, sorbitol, TMAO (trimethylamine -N- oxide), trimethyl glycine, DMSO (dimethyl sulfoxide), triethylene glycol, ethylene glycol, methanol, ethanol, urea, acetamide, and a 8-mer DNA and the like.

  The reaction conditions when RecA, Rad51 or Rad52 is used as the RecA family recombinase of the present invention or a protein having recombination activity are described. The amount of DNA ligase added to the reaction solution is not particularly limited, and an optimal unit number of the DNA ligase to be used may be used. In addition, when RecA is used as the RecA family recombination enzyme, the DNA ligase activity tends to occur when the DNA ligase activity is excessive compared to RecA. It is desirable that it does not become. In addition, when it is desired to prepare a long linear double-stranded DNA (multimeric linear double-stranded DNA), it is desirable to increase the amount of RecA relative to the amount of DNA ligase (see FIG. 7 and the like). ).

When RecA is used as the RecA family recombinase of the present invention, the amount of RecA added to the reaction solution is a molar ratio when the amount of nucleotides constituting the linear double-stranded DNA is 1 mol, and RecA is 0.01 to 0.1 is preferable, 0.02 to 0.05 is more preferable, and 0.04 is most preferable. In particular, in the presence of a molecule having a molecular crowding effect such as PEG and ATP, about 0.04 mol of RecA is appropriate for 1 mol of nucleotide constituting the linear double-stranded DNA, and the amount of RecA is Beyond this, the reaction efficiency tends to decrease. In addition, it is desirable not to add ATP under conditions where no molecules having a molecular crowding effect such as PEG are added. For example, in the presence of 1.3 mM ATP, ligation of linear double-stranded DNA is completely inhibited. Is done.
It should be noted that in this specification, the concentration of DNA is expressed as the concentration of nucleotides constituting DNA, and is not the molar concentration of linear double-stranded DNA itself.
On the other hand, when adding a molecule having a molecular crowding effect, such as PEG, ATP is added, for example, about 1 to 3 mM, and preferably about 1 to 2 mM. Further, a molecule having a molecular crowding effect such as PEG may be added, for example, about 1.0% to 10% (w / v), preferably about 2.5% to 5.0 (w / v) when PEG6000 is taken as an example. . Of course, the optimum concentration and the like can be determined as appropriate by those skilled in the art depending on the molecule having a molecular crowding effect to be used, and thus is not particularly limited to the above values.
In the presence of ATP and a molecule having a molecular crowding effect such as PEG, the ligation of linear double-stranded DNA by RecA proceeds very efficiently (for example, linear double-stranded DNA efficiently in a short reaction time). ) And suppress cyclization).
In addition, when adding MgCl ₂ during the reaction, conditions of 4 mM or more, more preferably 5 mM or more, and even more preferably 7 mM or more may be used.

When Rad51 is used as the RecA family recombination enzyme of the present invention, in order to promote ligation between linear double-stranded DNAs and to suppress circularization of the linear double-stranded DNA, during the reaction, The amount of Rad51 to be added is about 0.042 to 0.167, preferably about 0.083 to 0.167 in terms of molar ratio when the nucleotide constituting the linear double-stranded DNA is 1 mole (3 to 3 per Rad51 of one molecule). 6bp (base pair)). About ATP, it is better to add, and the concentration of ATP in this case is, for example, about 1 to 3 mM, and preferably about 1 to 2 mM. In addition, when adding molecules having a molecular crowding effect such as PEG, taking PEG6000 as an example, for example, 1.0% to 10% (w / v), preferably 2.5% to 5.0 (w / v) However, the optimum concentration and the like can be appropriately determined by those skilled in the art depending on the molecule having a molecular crowding effect to be used, and is not particularly limited to the above numerical values. In addition, when adding MgCl ₂ during the reaction, it is preferable to use conditions of 7 mM or more.

When Rad52 is used as the protein having recombination activity of the present invention, in order to promote the binding between linear double-stranded DNAs and to suppress the circularization of the linear double-stranded DNA, The amount of Rad52 added to is preferably 0.033 or more in terms of a molar ratio when the amount of nucleotide constituting the linear double-stranded DNA is 1 mol (Rad52 is 1 molecule or more with respect to 15 bp (base pair)). ATP may or may not be added, and the addition or non-addition of ATP may be determined depending on the ATP requirement of the DNA ligase used. In addition, when adding molecules having a molecular crowding effect such as PEG, taking PEG6000 as an example, for example, 1.0% to 10% (w / v), preferably 2.5% to 5.0 (w / v) However, the optimum concentration and the like can be appropriately determined by those skilled in the art depending on the molecule having a molecular crowding effect to be used, and is not particularly limited to the above numerical values. In addition, when adding MgCl ₂ during the reaction, it is preferable to use conditions of 7 mM or more.

  In carrying out the method of the present invention, the reaction temperature, time and the like can be appropriately selected by those skilled in the art in consideration of the characteristics of the DNA ligase used. In the case of T4 ligase or E. coli ligase, it is, for example, 35 ° C. to 40 ° C., preferably about 37 ° C. and about several minutes to several hours. The order of addition to the reaction solution is not particularly limited. For example, RecA family recombination enzyme, linear double-stranded DNA, and DNA ligase are added in this order, and each is added and incubated for several minutes to several hours. Also good.

  The RecA family recombination enzyme and the protein having recombination activity may be obtained from a natural source or may be obtained as a recombinant. In order to obtain a recombinant, a recombinant vector is required to express a RecA family recombinase or a protein having recombination activity. A nucleic acid encoding a RecA family recombinase or a protein having recombination activity is inserted into the recombinant vector so as to allow expression. Here, “expressible” means that the RecA family recombination enzyme having a desired amino acid sequence or a nucleic acid encoding such a recombination activity protein is combined with an open reading frame for expression. It is a state inserted into the vector and constructed in the vector so that other necessary components such as a promoter and a selectable marker function.

  Here, among nucleic acids encoding RecA family recombinase or a protein having recombination activity, each nucleic acid encoding RecA, Rad51 or Rad52 consists of a base sequence represented by SEQ ID NO: 2, 4 or 6, respectively. A nucleic acid and a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 2, 4 or 6, respectively, are hybridized under stringent conditions and are linear duplexes by DNA ligase Also included are nucleic acids that encode proteins that have the activity of promoting terminal binding between DNA molecules. The DNA capable of hybridizing under stringent conditions with a nucleic acid containing the base sequence represented by SEQ ID NO: 2, 4 or 6 is preferably about 70% or more with the base sequence represented by SEQ ID NO: 2, 4 or 6. More preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, and most preferably, a nucleic acid containing a nucleotide sequence having a polynucleotide sequence homology of about 99%.

Here, stringent conditions are conditions for hybridization that are easily determined by those skilled in the art, and are generally empirical conditions that depend on probe length, washing temperature, and salt concentration. is there. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability of the nucleic acid to reanneal under conditions where the complementary strand is slightly below its melting point.
Specifically, for example, as a low stringency condition, in a filter washing step after hybridization, washing is performed in a 0.1 × SSC, 0.1% SDS solution at a temperature of 37 ° C. to 42 ° C. Can be raised. Examples of highly stringent conditions include washing in 65 ° C., 5 × SSC and 0.1% SDS in the washing step. By making the stringent conditions higher, a highly homologous polynucleotide can be obtained.

  A nucleic acid encoding a RecA family recombination enzyme or a protein having recombination activity can be obtained from various cells according to a conventional method. Alternatively, the nucleic acid sequence information of a known RecA family recombination enzyme or a protein having recombination activity is obtained from a database or the like, and based on the sequence information, a RecA family recombination enzyme derived from a desired species or It is also possible to obtain a nucleic acid encoding a protein having recombination activity. Cloning of a nucleic acid encoding a RecA family recombination enzyme or a protein having recombination activity can be obtained by a screening method common in the art.

  Examples of vectors that can be used for expression of RecA family recombinase or proteins having recombination activity include plasmid DNA, phage DNA, and the like. As plasmid DNA, plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pCBD-C, pET15b etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194 etc.), yeast-derived (For example, YEp13, YEp24, YCp50, YIp30, etc.) and the like, and phage DNA include λ phage. Furthermore, animal viruses such as retroviruses and vaccinia viruses, and insect virus vectors such as baculoviruses and togaviruses can also be used.

The promoter used for expression of RecA family recombinase or a protein having recombination activity is not particularly limited as long as it is an appropriate promoter corresponding to the host used for gene expression.
For example, when animal cells are used as the host, SRα promoter, CMV promoter, SV40 promoter, LTR promoter, HSV-TK promoter, EF-1α promoter and the like can be mentioned.
When the host is Escherichia coli, the tac promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, etc., and when the host is Bacillus subtilis, the SPO1 promoter, SPO2 promoter, penP promoter Etc.
When the host is yeast, examples include PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, GAL promoter and the like.
When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the RecA family recombinase or a protein having recombination activity, the recombinant vector for expression of the RecA family recombination enzyme or the protein having recombination activity includes a selection marker, A terminator, enhancer, splicing signal, poly A addition signal, ribosome binding sequence (SD sequence), SV40 origin of replication (SV40ori) and the like can be linked.
The selection markers include, but are not limited to, hygromycin resistance marker ^(Hyg r), dihydrofolate reductase gene ^(DHF r), the ampicillin resistance gene ^(Amp r), the kanamycin resistance gene ^(Kan r), neomycin resistance gene (Neo ^r , G418) and the like can be used.
In addition, for the purpose of facilitating the isolation and purification of the recombinant protein, a tag sequence for purification, such as a His tag or HA tag, on the N-terminal side, C-terminal side, etc. , GST, etc. can also be fused.
When the host is Escherichia coli, alkaline phosphatase signal, OmpA signal, etc. can be used. When the host is Bacillus subtilis, α-amylase signal sequence, subtilis signal sequence, etc. can be used. In some cases, an α-factor signal sequence, an invertase signal sequence, and the like can be used. When the host is an animal cell, for example, an insulin signal sequence, an α-interferon signal sequence, and the like can be used.

  In order to express a RecA family recombination enzyme or a protein having recombination activity, it is necessary to transform an expression vector into which a nucleic acid encoding them is inserted into an appropriate host cell. The host for transformation is not particularly limited as long as it can express RecA family recombinase or a protein having recombination activity. For example, Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, Pseudomonas putida such as Pseudomonas, Rhizobium meliloti and other bacteria belonging to Rhizobium such as Rhizobium meliloti, Yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), or Sf9, Sf21 Insect cells such as

  As a method for introducing a recombinant vector into E. coli, a method using calcium ions, an electroporation method, or the like can be used. As a method for introducing a recombinant vector into yeast, an electroporation method, a spheroplast method, a lithium acetate method, or the like can be used. Examples of the method for introducing a recombinant vector into animal cells or animal cells include the DEAE dextran method, the electroporation method, the calcium phosphate method, and the method using a cationic lipid.

A RecA family recombinant enzyme or a protein having recombinant activity can be produced by culturing a transformant, expressing the enzyme, and isolating the enzyme from the culture of the transformant. “Culture” means any of culture supernatant, cultured cells or cultured cells, or disrupted cells or cells.
As a medium for cultivating transformants using microorganisms such as Escherichia coli and yeast as a host, the medium contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the microorganisms, and the transformants are efficiently cultured. Any natural or synthetic medium may be used as long as it can be used. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

  Culturing is performed under conditions suitable for the host cell. For example, as the medium for culturing Escherichia coli, LB medium, M9 medium and the like are preferable. Agents such as isopropyl-1-thio-β-D-galactoside, 3β-indolylacrylic acid can be added to make the promoter work efficiently if desired. In the case of Escherichia coli, the culture is usually carried out at about 15 to 37 ° C. for about 3 to 24 hours, and if necessary, aeration or stirring can be added. When the host is Bacillus subtilis, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and agitation can be added as necessary.

  Examples of the medium for culturing yeast include SD medium and YPD medium. The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary. When culturing a transformant whose host is an insect cell or an insect, examples of the medium include a grace insect medium containing bovine serum. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.

  When cultivating a transformant whose host is an animal cell, for example, a MEM medium, DMEM medium, RPMI-1640 medium or the like containing about 5 to 20% fetal calf serum is used as the medium. The pH is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary. Separation and purification of a RecA family recombinase or a protein having recombination activity from the culture can be performed, for example, by the following method.

  When extracting RecA family recombinase or a protein having recombination activity from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer solution, and subjected to ultrasound. For example, a method of obtaining a crude extract of RecA family recombinant enzyme or a protein having recombinant activity by centrifuging or filtering after lysozyme and / or freeze-thawing is used as appropriate. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. When RecA family recombinase or a protein having recombination activity is secreted into the culture solution, after completion of the culture, the cells or cells and the supernatant are separated by a method known per se, and the supernatant is collected. . Purification of the RecA family recombinase or protein having recombination activity contained in the culture supernatant or extract thus obtained can be performed by appropriately combining known separation and purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation methods, dialysis methods, ultrafiltration methods, gel filtration methods, and mainly using differences in molecular weight such as SDS-PAGE. Method, method utilizing the difference in charge such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography, isoelectric point A method using the difference in isoelectric point such as electrophoresis is used.

  The present invention also includes a kit comprising a RecA family recombinase or a protein having recombination activity, or an expression vector of a RecA family recombination enzyme or a protein having recombination activity. A kit for preparing double-stranded DNA in which double-stranded DNAs are connected in series is included. The kit of the present invention includes a RecA family recombinase or a protein having recombination activity, or an expression vector of the RecA family recombinase, as well as buffers and molecular crowding effects necessary for carrying out the method of the present invention. Molecules (eg PEG6000) and instructions for use may be included.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.

1. experimental method.
1-1. Restriction enzyme treatment.
The reaction conditions of restriction enzymes, PstI (Takara Bio Inc.), HindIII (Takara Bio Inc.) and HincII (Takara Bio Inc.) used in this example are as shown in Table 1 below. “BufferM” is a buffer attached at the time of purchase of each restriction enzyme.
The restriction enzyme treatment reaction was carried out at 37 ° C. for 15 minutes, and then treated with phenol / chloroform (phenol: chloroform: isoamyl alcohol = 25: 24: 1), followed by ethanol precipitation to recover DNA. Thereafter, the recovered DNA was dissolved in TE _0.1 buffer (10 mM TrisHCl (pH 8.0), 0.1 mM EDTA), and further dialyzed against TE _0.1 buffer.

1-2. DNA ligase reaction in the presence of RecA family recombinase.
The reaction conditions are as follows.
(Total volume 20μl)
MgCl ₂ : 7mM
DTT: 1.8mM
EDTA 0.1mM
TrisHCl: 31 mM (pH 7.5)
Above, buffer conditions. To this, RecA family recombination enzyme, linear double-stranded DNA, T4 ligase or E. coli ligase (Takara Bio Inc.) was added.
Further, the reaction was carried out under the conditions of adding or not adding the following.
± RecA: 2 μM, ± Rad51: 0.5 to 0.75 μM, ± Rad52: 0.4 μM
± NAD: 100 μM (for E. coli ligase)
± ATP: 1.3mM (T4 ligase)
± PEG6000: 0 to 10% (w / v)

The reaction was performed as shown in Table 2 below.
Specifically, RecA family recombinase was added to the above buffer, incubated at 37 ° C. for 3 minutes, then DNA was added, and incubated at 37 ° C. for 5 minutes (in Rad51 or Rad52, the above buffer with added DNA) Rad51 or Rad52 was added on ice and incubated at 37 ° C. for 5 minutes). Then, add DNA ligase and react at 37 ° C for 40 minutes. Add 1% SDS, 50μg / ml Proteinase K (Pro K) (final concentration) to the reaction product, and incubate at 37 ° C for 10 minutes. Stopped. Thereafter, the reaction product was electrophoresed on a 0.7% agarose gel. When ethidium bromide (EtBr) was added to the agarose gel, the final concentration was 0.06 μM.

2. result.
2-1. Review of conventional technology.
First, Non-Patent Document 1 and Non-Patent Document 2 disclose that ligation of heavy chain DNA is promoted in a straight chain in the presence of a molecule having a molecular crowding effect such as PEG. Therefore, the binding reaction of linear double-stranded DNA in the presence of PEG was examined.
A pUC119 double-stranded DNA (6 μM) having a 3 ′ protruding single-strand portion cleaved with PstI was reacted with NAD-dependent E. coli DNA ligase (Takara, 4U / 20 μL) at 37 ° C. for 40 minutes. When PEG6000 was added, DNA multimer formation by DNA ligase was promoted according to the amount. In the gel electrophoresis of the reaction product without Etidium bromide (EtBr), monomer (1L, 1C), dimer (2L, 2C), trimer (3L, 3C), tetramer (4L, 4C), pentamer (5L, 5C), hexamer or higher (>6L,> 6C) band-like signals were observed one by one (FIG. 1A). In contrast, when 0.06 μM EtBr is added to the agarose gel (B), monomer (1L), dimer (2L), trimer (3L), tetramer (4L), pentamer In addition to the signals of linear double-stranded DNA of hexamer or more, another signal was observed under each. These bands are signals of the respective circular double-stranded DNA (1C, monomer; 2C, dimer; 3C, trimer (overlapping with 2L); 4C, tetramer) (Figure 1B). In the leftmost lane, linear double-stranded DNA (1L) used as a reaction substrate is migrated. Only in this lane (the lane in which only linear double-stranded DNA is migrated), there is only one signal regardless of the presence or absence of EtBr.
From the above results, it is clear that it is difficult to eliminate the circularization of linear DNA in the ligation reaction of linear double-stranded DNA in the presence of only molecules having a molecular crowding effect such as PEG. is there.

Next, in Non-Patent Document 3, only the intermolecular binding by T4 DNA ligase is promoted by adding a high concentration of monovalent cation (in the case of NaCl, 150-200 mM) in the presence of PEG6000. Since this is reported, this point was examined.
PUC119 double-stranded DNA (6 μM) that has been cut with Pst1 and cleaved with PstI is added with the indicated concentrations of NaCl and PEG6000 of 3.8, 7.5, and 11.3% for each concentration of NaCl. T4 DNA ligase (20 U / 20 μL) was reacted at 37 ° C. for 40 minutes. The gel electrophoresis of the reaction product was performed without Etidium bromide (FIG. 2A) or in the presence of 0.06 μM Etidium bromide (FIG. 2B). As a result, it was clarified that the circular DNA dimer signal (2C) remained even in the reaction in the presence of 150 mM NaCl, 11.3% polyethylene glycol, 200 mM NaCl, 7.5% polyethylene glycol. It became clear that it was difficult to eliminate.
Based on the above state of the prior art, examples of the present invention will be described below.

2-2. Promotion of ligation of linear double-stranded DNA by RecA.
First, we investigated the ligation-promoting reaction of linear double-stranded DNA by RecA, a RecA family recombination enzyme.
(1) Promotion of ligation reaction of linear double-stranded DNA by RecA when PEG6000 is not added.
PUC119 linear double-stranded DNA (6 μM) that is cleaved with PstI and has a 3 ′ protruding single-stranded portion of 4 bases complementary at both ends is ligated with NAD-dependent Escherichia coli DNA ligase (Takara, 6U / 20 μL) The indicated concentration of RecA was added to the reaction (37 ° C., 40 minutes) (FIG. 3, RecA). In the absence of ATP, 2-4 μM RecA promoted the formation of linear DNA multimers and completely suppressed the formation of circular monomers (FIG. 3). These enhancements and suppressions were almost completely inhibited by 1.3 mM ATP (FIG. 3). In addition, excessive amounts of RecA did not promote the formation of linear DNA multimers. In addition, 0.06 μM Ethidium bromide was added to the electrophoresis gel in order to separate the circular double-stranded DNA of the same size as the linear double-stranded DNA of the substrate. Compared with FIG. 4, the formation of cyclic trimer (3C) and tetramer (4C) is slightly observed even under the condition of promoting multimer formation by RecA.

(2) Promotion of ligation reaction of linear double-stranded DNA by RecA when PEG6000 is added.
In the presence of the amount of PEG6000 shown in FIGS. 4A and B, a 4-base 5′-protruding single-stranded portion produced by digestion with HindIII by NAD-dependent E. coli DNA ligase (Takara, indicated amount / 20 μL) pUC119 linear double-stranded DNA (6 μM) (FIG. 4A), or pUC119 linear double-stranded DNA having a 3 base protruding single-stranded portion of 4 bases complementary to both ends made by cutting with PstI (6 μM) (FIG. 4B) was added to the reaction (37 ° C., 40 minutes) with 2 μM RecA. In the presence of PEG6000, RecA promoted DNA ligase multimer formation reaction only when ATP (1.3 mM) was present, and the formation of cyclic monomers and cyclic multimers was completely inhibited (linear monomer). No signal of cyclic multimer is detected between 1L, 2L, 3L, 4L, and 5L of linear monomer / multimer signal under 1L signal. This promotion occurs even with an excess of RecA.
In addition, 0.06 μM Ethidium bromide was added to the electrophoresis gel in order to separate the circular double-stranded DNA of the same size as the linear double-stranded DNA of the substrate. In the absence of PEG, enhancement of multimer formation by RecA is seen only in the absence of ATP (see FIG. 3).

(3) Examination of the effects of Mg ²⁺ .
Next, the influence of Mg ²⁺ present in the reaction solution was examined.
PUC119 linear double-stranded DNA cleaved with PstI in the presence of MgCl ₂ and NAD-dependent E. coli DNA ligase in the amounts shown in FIG. 5 and in the presence of 3.75% (W / V) PEG6000 (6 2 μM RecA, or 2 μM RecA and 1.3 mM ATP was added to the reaction (37 ° C., 40 minutes). In addition, since the difference in Mg ²⁺ concentration is thought to affect the activity of DNA ligase, for comparison of the degree of activity when RecA or the like is added, so that the activity of DNA ligase alone is the same, The amount of DNA ligase was adjusted for each MgCl ₂ concentration condition shown in FIG. 5 (DNA ligase; 3.5-7 μM). In PEG + RecA (without ATP), Mg ²⁺ concentration had no effect on the degree of ligation promotion of linear double-stranded DNA, but in PEG + RecA + ATP, 1 mM MgCl ₂ was linear. Binding between molecules of double-stranded DNA was completely inhibited.

(4) Examination of influence by reaction time.
The incubation time for ligating linear double stranded DNA was examined.
For ligation of linear double-stranded DNA using NAD-dependent Escherichia coli DNA ligase, incubate at 37 ° C for 40 minutes with PEG alone, PEG and RecA added (no ATP) Depending on the reaction time, unreacted linear double-stranded DNA decreased, resulting in DNA dimers and larger multimers (FIG. 6, upper figure and middle figure).
On the other hand, in the presence of PEG, RecA and ATP, the amount of DNA ligase used can be reduced to PEG alone, or to 1/4 of the amount of DNA ligase used under the conditions of PEG + RecA. Ligation of linear double-stranded DNA was promoted to the same extent as the two conditions (PEG only, PEG + RecA). Furthermore, under the condition of PEG + RecA + ATP, multimeric linear double-stranded DNA was generated at a stretch in about 2 minutes after the start of the reaction. From this result, it was found that, when RecA was added in the presence of PEG and ATP and a DNA ligation reaction was performed by DNA ligase, a multimer of linear double-stranded DNA was efficiently produced.

(5) Relationship between RecA and DNA concentration (the concentration of nucleotides constituting DNA)
In the presence of PEG6000 and ATP, the reaction time was 40 minutes, and the ligation reaction was examined when the concentration of linear double-stranded DNA and the concentration of RecA were changed (FIG. 7). From Fig. 7, it can be seen that when 1 molecule of RecA is added per 12 bp of DNA (base pair) (24 nucleotides) as the required amount of RecA when the amount of DNA is changed, almost no unreacted DNA disappears. I understand. When PEG6000 and ATP are present, the optimal condition is that there is one RecA molecule per 12 bp of DNA (base pair) (24 nucleotides). If there is too much RecA, the efficiency of the reaction will be reduced. It became clear.

2-3. Promotion of ligation reaction of linear double-stranded DNA by Rad51.
(1) Examination of Rad51-promoting effect of linear double-stranded DNA ligation reaction
PUC119 linear double-stranded DNA (6 μM) cleaved with PstI was ligated with ATP-dependent T4 DNA ligase (4U, Takara Bio Inc.) (37 ° C, 40 minutes) to obtain 1.3 mM ATP. The indicated concentration of Rad51 was added in the presence, in the presence of 7 mM MgCl _{2, in the} presence or absence of 3.75% PEG. As a result, it was found that by adding Rad51, it was possible to prevent binding within the linear double-stranded DNA molecule by T4 DNA ligase, that is, circularization (FIG. 8, upper and lower figures). However, when the amount of Rad51 was increased, it was also clarified that the binding between the molecules of the linear double-stranded DNA, that is, the ligation reaction of the linear double-stranded DNA was suppressed (FIG. 8 middle diagram). ). Therefore, it seems appropriate to add 0.5 to 1.0 μM of Rad51 per 6 μM of nucleotides constituting the linear double-stranded DNA.

(2) Effect of PEG6000 on the ligation promoting reaction of linear double-stranded DNA in the presence of Rad51.
PUC119 linear double-stranded DNA cleaved with PstI (nucleotide concentration: 6 μM) was ligated with NAD-dependent E. coli DNA ligase (37 ° C, 40 min) in the presence or absence of 1.3 mM ATP. 0.5 μM Rad51 was added in the presence of 7 mM MgCl ₂ and the indicated concentration of PEG6000. As a result, it was found that the amount of PEG of about 2.5 to 5.0% (w / v) was in the optimum range (FIG. 9).

2-4. Promotion of ligation reaction of linear double-stranded DNA by Rad52.
PUC119 linear double-stranded DNA (nucleotide concentration 6 μM) cleaved with PstI is added to ATP-dependent T4 DNA ligase (in this case, ATP is added to 1.3 mM), or NAD-dependent Escherichia coli DNA ligase (In this case, NAD is added so as to be 0.1 mM) In the presence of 7 mM MgCl ₂ , the indicated concentration of Rad52 was added to the reaction (37 ° C., 40 minutes). As a result, by adding Rad52, the ligation between the linear double-stranded DNA molecules by T4 DNA ligase and E. coli DNA ligase was significantly promoted, and the circularization within the linear double-stranded DNA molecule was apparent. It was suppressed (the upper figure of FIG. 10, the center figure, and the lower figure).

  The present invention relates to a technique that makes it possible to efficiently prepare a DNA construct having a desired sequence in genetic manipulation such as gene cloning. Therefore, the use of the present invention is expected to improve the working efficiency in all fields using genetic manipulation.

Claims (12)

  Promoting end-to-end binding between linear double-stranded DNA molecules by incubating multiple linear double-stranded DNAs with DNA ligase in the presence of RecA family recombinase or a protein with recombination activity, And a method of producing a double-stranded DNA in which a plurality of linear double-stranded DNAs are connected in series by suppressing binding at both ends in the linear double-stranded DNA molecule.   The method according to claim 1, wherein the end to which the linear double-stranded DNA molecule is bonded is a sticky end.   The method according to claim 1, wherein the end to which the linear double-stranded DNA molecule is bound is a blunt end.   The method according to any one of claims 1 to 3, wherein the RecA family recombination enzyme is selected from the group consisting of RecA and Rad51, and the protein having recombination activity is Rad52.   The method according to claim 4, wherein the RecA family recombinase is RecA.   6. The method according to claim 5, wherein the incubation is performed in the absence of ATP.   6. The method according to claim 5, further comprising incubating under conditions to which ATP and a molecule having a molecular crowding effect are added.   The method according to claim 1, wherein the RecA family recombination enzyme is Rad 51 and incubated in the presence of ATP.   The method according to claim 1, wherein the protein having recombination activity is Rad52.   Furthermore, it incubates on the conditions which added the molecule | numerator which has a molecular crowding effect, The method of Claim 8 or 9 characterized by the above-mentioned.   The method according to claim 7 or 10, wherein the molecule having a molecular crowding effect is polyethylene glycol.   RecA family recombination enzyme or a protein having recombination activity, or a kit containing an expression vector of RecA family recombination enzyme or a protein having recombination activity, and a plurality of linear double-stranded DNAs in series A kit for preparing double-stranded DNA linked to.