WO2001025425A1 - NUCLEAR LOCALIZED RecQ5 TYPE DNA HELICASE - Google Patents

Abstract

Novel human RecQ5 type DNA helicases β and η and genes encoding the same are isolated. It is clarified that three RecQ5 type helicases involving the publicly known RecQ type DNA helicase (α) are formed as the result of alternative splicing. Among all, the RecQ5 type DNA helicase β has a novel characteristic of being localized in nucleus. The isoform β provided herein possibly relates closely to diseases in association with chromosomal unstability.

Description

 Description Nuclear Localization R e c Q Type 5 DNA Helicase

TECHNICAL FIELD The present invention relates to a nuclear localization isoform of RecQ5 helicopter, a gene encoding the same, and uses thereof.

Landscape technology

DNA helicase (EC 3.6.1.-) is an important enzyme that acts on various biological reactions involving DNA in vivo or in microbial cells, and there are many types of them. The existence of multiple types of DNA lipase, especially in multicellular organisms, is easy considering that DNA-related reactions are diverse, including cell replication and proliferation, individual development and growth, and life support. Can be guessed. Generally, D'A helicase is also called a DNA unwinding enzyme, and is known to have an action of unraveling double-stranded DNA into single-stranded DNA. It is believed that the energy required for this effect is obtained by hydrolysis of ATP. In fact, it has been suggested that there are at least five types of biochemical reactions catalyzed by DNA helicase at the cellular level:

DNA replication, repairing, transcription,

DNA segregation and Recombination Among the numerous DNA helicases, the recently discovered D. \ A helicase of the RecQ type has attracted attention in view of its association with human disease and aging. RecQ-type DNA helicase refers to a helicinase that has a helicinase domain that exhibits homology of about 40% or more at the amino acid level with the helicase domain of the E. coli recQ gene. It is the name given. RecQ-type helicase was discovered in E. coli by Nakayama et al. (Mol. Gen. Gen et., 195, pp474-480, 1984). Next, a gene encoding a protein with high homology to this helicase was found in yeast and human cancer cells, and each of them was identified as sgsl (Ganglof et al., Mol. Cell. Biol. 14, pp839 1-8398 ( 1994)) and RecQl (Seki et al., Nuc. Acida Res. 22, pp4566-4573 (1994)). In Bloom's syndrome, a disease that causes a variety of cancers at a young age, BLM helicinase has been identified as the causative gene (Cell, 83, pp655-666, 1995). In addition, it has been revealed that the WRN helicase mutation and loss of nuclear translocation activity is a direct cause of the inherited disease called Werner's syndrome that causes premature aging and abnormal cancer ( Science, 272, pp258-262.1996). Helicases belonging to this family include E. coli RecQ helicase, which was first found in single-celled organisms such as E. coli and yeast, and sgsl, which was found as its homologue in yeast. It is only. On the other hand, in humans composed of multiple cells, the above-mentioned disease-related BLM helicase ゃ WRN helicase and the sum of Rec Q1 helicase that have not yet been found to be associated with the disease Three types have been known so far. In addition, the present inventors have reported two types of RecQ-type DXA helicases, RecQ4 and RecQ5 (Genomics 54, 443-452, 1998). RecQ4 and RecQ5 found by the present inventors are characteristically different from other RecQ-type DNA helicases in that, as a result of homology search, a known amino acid sequence like a nuclear translocation signal cannot be found near the C-terminus. there were. Furthermore, the present inventor Found that mutations in the RecQ4 gene cause Rosmund-Thomson syndrome (S. Ki tao et al., 1998, Genomi cs, 54, pp443-452; S. Ki tao et al., 1999). , Nat. Genet. 22: 82-84; WO99 / 05284; Ross Mundo 'Thomson Syndrome is a chromosomal instability disorder with multiple cancers that shows symptoms similar to Bloom and Werner syndromes. Against this background, the ability to elucidate the function of the RecQ family and the mechanism of chromosome stabilization has become an important key to elucidating the symptoms of these RecQ gene-related diseases, including multiple cancers. DISCLOSURE OF THE INVENTION An object of the present invention is to isolate a novel RecQ-type DXA helicase. More specifically, it is an object of the present invention to provide a nuclear localization isoform of human RecQ5 DNA helicopter, a polynucleotide encoding the same, and uses thereof. The present inventors have analyzed the structure and expression of the previously reported RecQ4 and RecQ5 type DNA helicases. They also noticed that observations of intracellular localization using recombinant RecQ5 helicase show localization of RecQ5 helicase to the cytoplasm. Many previously reported RecQ-type helicases, such as RecQl, BLM, or WRN, have been localized to the nucleus and have been suggested to function on chromosomal DNA. Even in RecQ4-type DNA helicase, for which no nuclear translocation signal could be found by homology search, localization to the nucleus was observed by similar experiments. Based on such findings, the present inventors predicted the presence of a nuclear-localized isoform in RecQ5-type DNA helicase. To support this prediction, the present inventors screened a human cDNA library using RecQ5 cDNA as a probe, and succeeded in isolating two RecQ5 isoforms. These isoforms of RecQ5 DNA helicase, including the previously reported RecQ5 DNA helicase, have been shown to be expression products resulting from alternative splicing. And One of these isoforms was found to be a novel RecQ5-type DNA-recycling DNA with nuclear localization, and the present invention was completed. That is, the present invention relates to the following nuclear-localized Recec5-type DNA helicase, its gene, and its use.

[1] The polynucleotide according to any one of the following (a) to (d):

 (a) a polynucleotide comprising a protein coding region having the nucleotide sequence described in the sequence;

(b) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2;

(c) an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids are modified by deletion, addition, insertion and / or substitution by another amino acid, and have helicase activity. A polynucleotide encoding a nuclear localization protein having the same.

(D) SEQ ID NO: A protein encoded by DN ^T A to Haiburidizu in DNA and scan Bok stringent conditions consisting of the described nucleotide sequence to 1, nuclear localization with helicopter force Ichize activity A polynucleotide encoding a sex protein.

 [2] A protein encoded by the polynucleotide according to [1].

 [3] A vector into which the polynucleotide according to [1] has been inserted.

 [4] A transformant carrying the polynucleotide of [1] or the vector of [3].

 [5] The method for producing the protein according to [2], comprising culturing the transformant according to [5] and collecting an expression product.

 [6] a polynucleotide comprising a polynucleotide comprising a nucleotide sequence corresponding to 1390 to 3703 in the nucleotide sequence of SEQ ID NO: 1 or a nucleotide having a nucleotide sequence complementary to a complementary strand thereof, at least 15 nucleotides in length Polynucleotide with.

 [7] The primer for polynucleotide synthesis according to [1], comprising the polynucleotide according to [6].

(8) The polynucleotide according to (1), comprising the polynucleotide according to (6). Probe for detecting

 [9] An antisense DNA against the polynucleotide or a part thereof according to [1].

 [10] a partial peptide consisting of an amino acid sequence corresponding to positions 411 to 991 in the amino acid sequence of SEQ ID NO: 2;

 [11] An antibody that recognizes a partial peptide consisting of an amino acid sequence corresponding to positions 411-1991 in the amino acid sequence of SEQ ID NO: 2.

 [12] immunology comprising a step of contacting the antibody of [11] with a protein consisting of the amino acid sequence of SEQ ID NO: 2 or a partial peptide thereof, and detecting an immunological reaction between the two. Measurement method.

 [13] A reagent for detecting a protein comprising the amino acid sequence of SEQ ID NO: 2 or a partial peptide thereof, comprising the antibody of [11].

 [14] A transgenic non-human vertebrate having an altered expression level of the polynucleotide according to [1].

 [15] The transgenic non-human vertebrate of [14], which is a knockout animal lacking the function of the polynucleotide of [1].

The isoform of RecQ5-type DNA polymerase provided by the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. The present inventors named the first reported RecQ5-type DNA helicopterase α, and the isoform of the RecQ5-type DNA helicopter according to the present invention as 3. Hereinafter, the three forms of RecQ5 DNA helicopter are described as RecQ5 β, and the α form as RecQ5. RecQ53 discovered for the first time by the present inventors was confirmed to have nuclear translocation activity and to be localized in the nucleus by an intracellular localization experiment using a recombinant. On the other hand, RecQ53 can be said to be a protein having novel characteristics, since the known RecQ53 is localized in the cytoplasm. Re of the present invention CQ5 / 3, like the known RecQ-type DNA helicase, contains, at the amino acid level, seven helicase 'motifs that are well conserved between E. coli, yeast and humans. Further, it was confirmed that the gene is present on the long arm of chromosome 17 of human, 17q25, similarly to RecQ5a in the genome. RecQ53 according to the present invention is a protein consisting of 991 amino acid residues, and its structure is as shown in FIG. In FIG. 3, the structure of RecQ5 / 3 of the present invention was compared with the structure of known RecQδα (410 amino acids) and the structure of RecQ5α (435 amino acids) confirmed together with RecQ5 / 3 of the present invention. The area shown by the line is a common part of the three isoforms. The area shown by the black box in this common area corresponds to the helicase domain. The shaded region is present only in RecQ5 / 3 and is homologous to the C-terminal region of E. coli RecQ. The region located further on the C-terminal side is a region specifically found in RecQ53, and as described later, a part of this region is presumed to be essential for the nuclear translocation activity of RecQ5 / 3. Was. From the results of genome analysis, these three isoforms were confirmed to be alternative splicing forms. Figure 2 shows the use of exons for each isoform. The three isoforms have in common exons 1 to 6, and for the large seventh exon including the 3 'UTR, RecQ5a has its entirety, while RecQ5) 3 has a portion on its 5' side. 7a, and RecQ5 uses the same 7a and 7b, which contains part of the 3 'UTR. RecQ53 also uses exons 8 to 19, which are not used in other isoforms.

RecQ5a (1-6) -7 / 3715nt.

RecQ5 / 3 (l-6) -7a- (8-19) / 3703m.

RecQ5r (l-6) -7a-7b / 1749nt.

The structure of the genome encoding the RecQ5 isoform is shown in FIG. FIG. 1 shows that the human RecQ5 gene is composed of 19 exons and 18 introns. In the figure, the length of the intron indicated by the broken line has not been determined. The protein of the present invention having the amino acid sequence of SEQ ID NO: 2 can be extracted from a human tissue that expresses this protein. Specifically, a protein is extracted from a tissue such as a human testis in which high expression is observed by a known method, and the RecQ5 of the present invention is extracted by a method such as immunoaffinity chromatography using an antibody against Recec5i3. 3) can be purified.

 The present invention also relates to an amino acid sequence represented by SEQ ID NO: 2, wherein one or more amino acids are modified by deletion, addition, insertion, and substitution with Z or another amino acid, and the helicase activity It relates to nuclear localized proteins with The nuclear localization protein means a protein having an activity of translocating to the nucleus. Therefore, for example, even when a part of the protein is distributed in the cytoplasm, if the protein is relatively strongly distributed in the nucleus, the protein is said to have nuclear localization.

 In the case of WRN helicase, which is considered to be localized in the nucleus, when the localization is observed in actual cultured cells, there is a case where localization to the nucleus is not recognized in a part of all cells. This is a phenomenon caused by the localization of WRN helicease in the cytoplasm in M-phase cells. In fact, the proportion of M-phase cells in cultured cells (5%; less than ~ 10%) corresponds to the proportion of cells in which nuclear localization of WRX helicase is not observed. Because WRN helicopter is not involved in nuclear division (chromosome segregation), it is thought to be in the cytoplasm during M phase. In the case of RecQ5 / 3 of the present invention, as in the case of WRN helicase, nuclear localization is observed in 90% or more of the cultured cells. Therefore, if nuclear localization is observed in more than 90% of the cells to be observed, the protein is said to be nuclear localized.

In the present invention, a nuclear-localized protein having a mutation in the amino acid sequence of SEQ ID NO: 2 and having helicase activity is referred to as a RecQ53 homolog. The RecQ53 homolog is a protein functionally equivalent to the human RecQ53. The RecQ5 i3 homolog consists of an amino acid sequence that has no significant homology to other known RecQ type D ^: A helicases. The significant homology in the present invention refers to the amino acid sequence shown in SEQ ID NO: 2. At least 50%, preferably 60%, more preferably 70% or more. Amino acid sequence homology is identified by BLAST (Altschul, SF, et al. 1990 J Mol Biol. 215: 403-410; Altschul, SF, et al. 1997 Nucleic Acids Res. 25: 3389-3402). Since the homology of the amino acid sequence of RecQ-type helicase in humans is usually 40% or less, the RecQ5 homolog of the present invention is clearly distinguished from known RecQ-type DNA helicase.

 In the RecQ53 protein according to the present invention, not only localization in the nucleus but also interaction with topoisomerase 3 and 33 were observed. Topoisomerase has been identified as a group of enzymes that have the ability to mitigate the twisting and entanglement of DNA molecules by cutting the DNA and to maintain the proper state. It is classified into type I and type I by its action. Topoisomerase 1 and topoisomerase 3 are known as type I topoisomerase, and topoisomerase 2 is known as type II topoisomerase. Among them, it has been shown that topoisomerase 3 may contribute to chromosome stabilization through interaction with sgsl in yeast. (Gangloff, S. et al. 1994 Mol Cell Biol. 14: 839, 8398). As described above, since yeast sgsl is a homolog of human RecQ, the interaction between RecQ-type helicase and toboisomerase 3 was confirmed in humans as well. This confirms that RecQ53 protein is closely related to chromosome stabilization in human cells.

 Therefore, the functionally equivalent protein of the RecQ5 / 3 protein of the present invention may be a protein localized in the nucleus having helicase activity and having an interaction with topoisomerase 3. desirable. The interaction between the RecQ53 homolog and topoisomerase-3 can be confirmed by detecting the binding between the two.

The number and location of amino acid mutations in the RecQ5 | 3 homolog are not limited as long as their functions are maintained. The number of mutations is typically within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 5% of all amino acids. For example, at least one amino acid sequence represented by SEQ ID NO: 2, preferably 1-1 About 0, more preferably 1 to 5 amino acids may be deleted. Alternatively, at least one, preferably about 1 to 10, and more preferably 1 to 5 amino acids can be added to the amino acid sequence represented by SEQ ID NO: 2. In addition, at least one, preferably about 1 to 10, and more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 can be substituted with another amino acid. These mutations may include not only one kind but also several mutations at the same time. Accordingly, proteins in which the first methionine (Met) in the amino acid sequence represented by SEQ ID NO: 2 has been deleted are also included in the proteins resulting from the change in the amino acid sequence. In addition, polymorphism can often be observed in the nucleotide sequence of genes encoding eukaryotic proteins such as RecQ-type DNA helicase. Polymorphism is a small-scale substitution of a nucleotide found in the nucleotide sequence of a gene, and the substitution usually has a small effect on the activity of the protein. The protein of the present invention also includes a protein in which a small variation in the nucleotide sequence or amino acid is caused by the polymorphism, if the protein has a ligase activity to nuclear-localized DNA.

 A homologue of a protein consisting of an amino acid sequence modified by deletion, addition, insertion and substitution with Z or another amino acid to the amino acid sequence of SEQ ID NO: 2, that is, a homologue of RecQ5i3 is, for example, It can be obtained by the method described.

The homolog of RecQ5 / 3 is isolated using a hybridization technique, a gene amplification technique, or the like well known to those skilled in the art. Specifically, the base sequence of the DNA encoding human RecQ5 | 3 was determined using hybridization technology (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wily & Sons Section 6.3-6.4). Based on the sequence (SEQ ID NO: 1) or a part thereof, DNA with high homology can be isolated. Obtaining a protein functionally equivalent to human RecQ53 from the obtained DNA can be performed by those skilled in the art. Thus, proteins encoded by DNA that hybridizes to DXA encoding human RecQ5; 3, Equivalent proteins are also included in the protein of the present invention. Examples of the organism from which the RecQ53 homolog according to the present invention is isolated by the hybridization technology include, in addition to humans, for example, rats, mice, rabbits, chickens, chickens, bushes, and shellfish. Alternatively, the RecQ5i3 homolog according to the present invention can be obtained not only in vertebrates but also in organisms such as yeast and C. elegans.

 The stringency of the hybridization for isolating the DNA encoding the RecQ5 / 3 homolog is generally "lxSS 0.1¾SDS, 37 ° C". As a stringent condition, the condition of “0.5xSSC, 0. \% SDS, 42 ° C” can be shown. As a more stringent condition, a condition of “0.2xSS 0.1¾SDS, 65 ° C” can be shown. As stringency becomes more stringent, isolation of DNA having higher homology to the probe sequence can be expected. Here, the above combinations of SS (:, SDS and temperature conditions are examples, and those skilled in the art will recognize other factors that determine the stringency of hybridization (eg, probe concentration, probe length, etc.). The same stringency as described above can be realized by appropriately combining the hybridization reaction time and the like.

 In general, the protein encoded by DXA isolated using such a hybridization technique has a high homology in the amino acid sequence to RecQ53 consisting of the amino acid sequence of SEQ ID NO: 2. High homology refers to sequence identity of at least 50% or more, preferably 60% or more, more preferably 70% or more. Amino acid sequence homology can be determined using the BLAST search algorithm. These nucleotide sequences constituting the DNA according to the present invention have high homology to the base sequence of RecQ53 consisting of SEQ ID NO: 1. In the present invention, high homology in the nucleotide sequence means at least 70% or more, preferably 80% or more, more preferably 90% or more sequence identity.

In addition, gene amplification technology (PCR) (Current protocols in Molecular Biology editor. Ausubel et al. (1987) Publish. John Wiley Sons Section 6.1-6.4) It can also be used to isolate DNA (or a fragment thereof) encoding the RecQ5 homolog. That is, when PCR is performed using a primer designed based on a part of SEQ ID NO: 1 (DNA encoding RecQ5 | 3 of the present invention), at least the region where the primer hybridizes is homologous to SEQ ID NO: 1. DNA with high potential is amplified. Based on the amplification products obtained, it is also possible to obtain the proteins encoded by them. The protein thus obtained may be composed of an amino acid sequence highly homologous to RecQ5 / 3 according to the present invention, similarly to the protein obtained by the hybridization technology. Is high.

 The DNA helicase activity of the protein obtained by modifying the amino acid sequence of SEQ ID NO: 2 can be confirmed by a known method ('. Suzuki et al., 1997, Nucleic Acids Res. 25: 2973-). 2978; MD Gray et al., 1997, Nature Genet. 17: 100-103). In addition, localization to the nucleus can be confirmed by expressing a gene encoding a RecQ53 homolog using a eukaryotic cell as a host and observing the intracellular localization. At this time, localization can be easily observed by fusing a marker protein such as EGFP to the gene encoding the RecQ5i3 homolog (T. Matsumoto et al., 1997). Nature Genet. 16: 335-336; X. Suzuki et al., 1997, Nucleic Acids Res. 25: 2973-2978). Alternatively, as shown in the examples, it is also possible to fuse an HA tag and detect it immunochemically with an anti-HA antibody.

Based on the disclosure of the present invention, a partial peptide of RecQ5i3 (or a homolog thereof) can be obtained. The partial peptide according to the present invention includes an antigen peptide for preparing an antibody. In order for the amino acid sequence constituting the partial peptide to be specific to RecQ5 (3, the amino acid sequence corresponding to positions 441 to 991 (C-terminal) in the amino acid sequence of SEQ ID NO: 2, or The number of amino acids constituting the partial peptide should be at least 7 amino acids, preferably at least 8 amino acids, more preferably at least 9 amino acids. A continuous amino acid sequence consisting of an acid sequence is desirable. As will be described later, this region contains an amino acid sequence necessary for the expression of nuclear translocation activity. For example, according to the analysis results of the present inventors, the region of 246 amino acids (positions 746 to 991 counted from the N-terminal side) located at the C-terminal side is necessary for the expression of nuclear translocation activity. Was suggested. A partial peptide consisting of an amino acid sequence selected from the amino acid sequences constituting this region is particularly desirable as an immunogen for obtaining an antibody to RecQ5 (3).

 The partial peptide according to the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase.

 In addition, the present invention relates to a polynucleotide encoding the protein of the present invention. In the present invention, polynucleotide means a molecule in which a large number of nucleotides are polymerized. The number of nucleotides to be polymerized is not particularly limited, but when the degree of polymerization is relatively low, it is also expressed as an oligonucleotide. The polynucleotide or oligonucleotide of the present invention can be naturally occurring or chemically synthesized. Alternatively, it may be synthesized by an enzymatic reaction such as PCR based on type III DNA. In the polynucleotide of the present invention, examples of the derivative of the polynucleotide include a structure that is made nuclease resistant by introducing a phosphorothioate bond, a polynucleotide into which a labeled molecule is introduced, and the like.

The form of the polynucleotide of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, and the like, in addition to cDNA. In addition, a polynucleotide having an arbitrary nucleotide sequence based on the degeneracy of the genetic code is included as long as it can encode the protein of the present invention. As described above, the polynucleotide of the present invention can be obtained by a hybridization technique using the full length or a part of the DNA base sequence (SEQ ID NO: 1) encoding human RecQ5 / 3 as a probe, It can be isolated by a PCR method or the like based on a primer synthesized based on the sequence. For example, human genomic DNA encoding RecQ53 can be obtained from 17q25, the long arm of chromosome 17. In the case of cDNA, for example, it can be obtained from a human sperm cDNA library. Furthermore, it has already been mentioned that homologues of other species of RecQ53 can be obtained.

 In addition, a mutation can be artificially introduced into the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1. Techniques for introducing mutations artificially are known (for example, site-directed mutagenesis (Current Protocols in Molecular Biology edit. Ausubel et al. (187) Publish. Jhon Wily Sons Section 8.1-8.5)). In addition, a point mutation introduction kit is also commercially available (for example, TAKARA LA PCR in virtro Mutagenesis Kit manufactured by Takara Shuzo Co., Ltd.).

 The DNA encoding RecQ5 / 3 (or a homolog thereof) according to the present invention can be used as a vector for expressing RecQ5 / 3 (or a homolog thereof) by incorporating it into an appropriate vector. Further, by transforming this vector into a host capable of expressing the same, the transformant according to the present invention can be obtained.

 The vector DXA for inserting a DNA fragment is not particularly limited as long as it can replicate in a host cell, and examples thereof include plasmid DNA and phage DNA. Examples of the plasmid DNA include plasmids pUC118 (manufactured by Takara Shuzo), pBluescript SK + (manufactured by Stratagene), and pGEM-T (manufactured by Promega). Examples of the phage DNA include M13mpl8 and M13mpl9. Can be

 When using plasmid DNA as vector DNA, for example, when inserting an EcoRI DNA fragment, digest the plasmid DNA with the restriction enzyme EcoRI (manufactured by NEB). Next, the DNA fragment and the cut vector DNA are mixed, and the resulting mixture is treated with, for example, T4 DNA ligase (Takara Shuzo) to obtain a recombinant vector.

The host is not particularly limited as long as it can express the gene of interest, and any of eukaryotic cells and prokaryotic cells can be used. As eukaryotic cells, yeast, Animal cells, such as COS cells and CHO cells, and insect cells. On the other hand, prokaryotic cells include bacteria such as Escherichia coli and Bacillus subtilis.

 When a bacterium such as Escherichia coli is used as a host, it is preferable that the recombinant vector of the present invention be capable of autonomous replication in the host and, at the same time, contain a promoter, the gene of the present invention, and a transcription termination sequence. .

 Examples of Escherichia coli include XL-Blue (manufactured by Stratagene) and] M109 (manufactured by Takara Shuzo). Examples of expression vectors include ρΒΤϊρ2. As the promoter, any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example, promoters derived from Escherichia coli or phage, such as the ρ promoter, the lac promoter, the PL promoter, the PR promoter, and the like, may be used.

 In the present invention, transformation is carried out, for example, by the method of ¾anahan [Techniques for Transformation of E. coli In DNA Cloning, Vol. 1, Glover, DM (ed.), Ppl09-136, IRL Press (1985)]. It can be carried out.

 When yeast is used as a host, examples of expression vectors include Yepl3 and Ycp50. Examples of the promoter overnight include the gal 1 promoter and gal 10 promoter overnight. Methods for introducing a recombinant vector into yeast include, for example, the electroporation method [Methods. Enzymol., 194, ppl82-187 (1990)] and the supheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)] and the lithium acetate method [J. Bacteriol., 153, 163-168 (1983)].

 When an animal cell is used as a host, pcDNAI, pcDNAI / Amp (Invitrogen) or the like is used as an expression vector. Methods for introducing the recombinant vector into animal cells include, for example, the elect-mouth poration method, the lipofection method, and the calcium phosphate precipitation method.

When an insect cell is used as a host, expression vectors such as pVL1392, pVLl393 (Invitrogen), pMBac (Stratagene), pBacPAK8Z9 (Clonte ch company) is used. Examples of a method for introducing a recombinant vector into insect cells include the calcium phosphate method, the ribofection method, and the electroporation method.

 The above-mentioned screening of the transformant is carried out by colony hybridization using a DXA fragment containing a part of the target gene as a probe, or 5 ′ primer (FP: forward) based on the base sequence of the target gene. primer), and then 3 ′ primers (RP; reverse primer) based on the base sequence of the complementary strand DNA, and the PCR method using these primers A colony (transformant) containing the desired gene can be selected and collected.

 Furthermore, by culturing the transformant of the present invention, RecQ5 of the present invention (or a homolog thereof) can be produced. The culture method is not particularly limited, but by adopting a liquid culture method, operations such as induction of expression, separation of cells, or isolation of an expression product can be easily performed. The method for culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host.

 As a medium for culturing a transformant using a microorganism such as Escherichia coli or yeast as a host, any of a natural medium and a synthetic medium can be used. Specifically, the medium is not particularly limited as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism and can efficiently culture the transformant.

 The cultivation is usually carried out under aerobic conditions such as shaking culture or aeration-agitation culture at 35 to 40 ° C, preferably around 371: 14 to 20 hours. During the culture period, the pH is adjusted to 7.2-1.4. During the culture, antibiotics such as ampicillin / tetracycline, and bimin, etc. can be added to the medium as needed.

When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the Lac promoter, use isopropyl-3-D-thiogalactovyranoside (IPTG), etc., and use 1 卬 promoter. When culturing a microorganism transformed in the same expression vector, indoleacrylic acid (IAA) or the like may be added to the medium.

 When culturing a transformant using an animal cell as a host, a commonly used RPMI-1640 medium, DMEM medium, or a medium obtained by adding fetal bovine serum or the like to such a medium is used. Culture is usually performed at 37 ° C in the presence of 5% C (^. During culture, antibiotics such as kinamicin and benicillin may be added to the medium as needed.

 Grace's Insect Medium (Grace, TC C. Nature, 195: 788, (1962)) shows 10% of the area for culturing transformants obtained using insect cells as a host.が One that is appropriately added with additives such as fetal serum. Adjust the pH of the culture medium to 6.6 to 7.0, and incubate at 25 for 1 to 7 days. Add aeration and / or agitation as necessary.

 After completion of the culture, a normal protein purification means can be used to collect the protein of the present invention from the culture (in the cell lysate, the culture, or the supernatant thereof). After culturing, when the protein of the present invention is produced intracellularly or intracellularly, the cells are destroyed by lysis treatment using an enzyme such as lysozyme, ultrasonic crushing, grinding, etc. The protein of the present invention is excreted outside the cells. Then, insoluble materials are removed by filtration or centrifugation to obtain a crude protein solution.

 On the other hand, when the protein of the present invention is produced extracellularly or extracellularly, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like to obtain a supernatant. Then, common biochemical methods used for the isolation and purification of proteins, such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, affinity chromatography, and electrophoresis, are used. The protein of the present invention can be isolated and purified from the above-mentioned cultures singly or in an appropriate combination.

Alternatively, if the RecQ53 protein of the present invention is expressed as a fusion protein with a binding ligand, purification by affinity chromatography becomes possible by combining the protein with a receptor for the binding ligand. For example, the histidine residue A method for purifying a fusion protein with a histidine tag consisting of a series of the above using a nickel column is known. A histidine tag-containing vector for this purpose is also commercially available. It is convenient to fuse the binding ligand and RecQ53 via a linker that can be cleaved with protease or the like, since the expression product adsorbed to affinity chromatography can be easily recovered.

 DM encoding RecQ5 i3 of the present invention can be used for gene therapy. RecQ53 of the present invention has nuclear translocation activity and can be said to be an enzyme capable of expressing chromosome stabilizing action in vivo, which can be said to be an essential function of DNA helicase. Therefore, by inoculating the vector of the present invention into which RecQ5β has been incorporated so that it can be expressed, a therapeutic effect on chromosome instability caused by RecQ5 deficiency or mutation can be expected. Examples of the vector into which the DNA encoding the RecQ5 i3 of the present invention can be introduced into a patient include a viral vector derived from a retrovirus or an adenovirus, and a non-viral vector using a ribosome or the like. Can be

 Further, the present invention provides an antibody against the protein of the present invention. The antibody of the present invention uses RecQ53 homologue containing RecQ5β or the aforementioned partial peptide of RecQ5 / 3 as an immunogen, and as a monoclonal antibody or a polyclonal antibody based on a known method. Obtainable. The monoclonal antibody according to the present invention can be obtained, for example, as follows.

 (1) Preparation of antigen

 The RecQ5 3 homologue containing RecQ5 β or a partial peptide thereof is dissolved in a buffer and then mixed with an adjuvant to prepare an immunogen. Examples of the adjuvant include commercially available complete Freund's adjuvant and incomplete Freund's adjuvant, and any of these may be mixed.

 (2) Collection of immune and antibody-producing cells

The above-mentioned immunogen is administered to a mammal, for example, a rat or a mouse. The immunological amount of the antigen is 10 to 500 ^ per animal at a time. The immunization site is mainly intravenous, subcutaneous, Intraperitoneally. The interval of immunization is not particularly limited, and immunization is performed 2 to 5 times, preferably 3 to 4 times at intervals of several days to several weeks, preferably at intervals of 1 to 3 weeks. Two to seven days, preferably four to five days after the last immunization date, a sufficient increase in the antibody titer is confirmed, and the antibody-producing cells are collected. Examples of the antibody-producing cells include B cells derived from spleen cells, lymph node cells, peripheral blood cells, etc., and B cells derived from spleen cells or local lymph node cells are preferred.

 (3) Cell fusion

As myeloma cells to be fused with the antibody-producing cells, commonly available cell lines of animals such as mice are used. The cell line used is drug-selective, cannot survive in a selective medium (HAT medium: containing hypoxanthine, aminopterin, thymine) in an unfused state, and is fused with antibody-producing cells. It is preferable that it has the property of being able to survive only. Specific examples of myeloma cells include mouse myeloma cell lines such as P3U-1 (Dainippon Pharmaceutical Co., Ltd.) and P3x63Ag8.653. Next, the myeloma cells are fused with the antibody-producing cells. For cell fusion, an equal volume of 10 ⁸ cells / ml of antibody-producing cells and 2 x 10 ⁵ cells / ml of myeloma cells in an animal cell culture medium such as serum-free DMEM or RPMI-1640 medium Mix and perform the fusion reaction in the presence of the fusion promoter. In order to promote cell fusion, polyethylene glycol having an average molecular weight of 1,500 dalton or the like can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (e.g., electoporation).

 (4) Selection and cloning of Hypri-Doma

Select the desired hybridoma from the cells after cell fusion. As a method, after appropriately diluting the cell suspension with, for example, RPMI-1640 medium containing fetal bovine serum, spread about 5 to 10 cells in a microtiter plate on a plate, and select each well. The medium is added, and the culture is performed after replacing the selection medium appropriately. As a result, cells that grow about 14 days after the start of culture in the selection medium can be obtained as hybridomas. Whether the target antibody is present in the culture supernatant of the growing hybridoma To screen. Screening for hybridomas may be performed according to a conventional method, and is not particularly limited. For example, a part of the culture supernatant contained in the gel grown as a hybridoma is collected, and the antibody is screened by ELISA or RIA. The fused cells for which production of the desired antibody has been confirmed are cloned by a limiting dilution method or the like, and finally, a hybridoma, which is a monoclonal antibody-producing cell, is established.

 (5) Collection of monoclonal antibodies

 As a method for collecting a monoclonal antibody from the established hybridoma, a normal cell culture method, an ascites formation method, or the like can be used.

In the cell culture method, hybridomas are cultured in animal cell culture medium such as RPMI-1640 medium, MEM medium or serum-free medium containing 10% fetal serum at normal culture conditions (for example, at 37 ° C, 5% CO2). ₂ concentrations) for 10 to 14 days, and antibodies can be obtained from the culture supernatant. In the case of the ascites formation method, about 5 × 10 ⁶ hybridomas are administered intraperitoneally to mammals and allogeneic animals derived from myeloma cells, and the hybridomas are grown in large quantities. Then, ascites is collected 1-2 weeks later.

 When antibody purification is required in the above antibody collection method, a known method such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, gel chromatography or the like is appropriately selected, or Purification can be performed by combining these.

 The antibody of the present invention can also be obtained as a polyclonal antibody based on, for example, the following method.

 (1) Preparation of antigen

 Prepare an immunogen similar to the immunogen for the monoclonal antibody.

 (2) Immunity

Normally, animals such as egrets, guinea pigs, goats, and sheep are used as animals. Taking egrets as an example, 100 xg to 500 g of polypeptide is usually placed on the egret footpad. Inject subcutaneously with complete Freund's adjuvant. Two weeks later, the same amount of antigen is mixed with incomplete Freund's adjuvant and injected intramuscularly. Two weeks later, intramuscular injection is repeated, and one week after the final immunization, blood is partially collected from the ear and the antibody titer is measured by EIA method or the like. If the antibody titer has reached the target value, collect whole blood. If the antibody titer is low, repeat intramuscular injection, and repeat immunization until the antibody titer reaches the target value. For the purification of antibodies from serum by ammonium sulfate fractionation, the method described in the section on monoclonal antibodies can be adopted.

 If all the molecules of RecQ53 are used as the immunogen, an antibody specific to RecQ53 can be obtained by performing an absorption operation using a region common to other RecQ5-type DNA helicases containing a helicase domain. Can be.

 Known mutations in the human RecQ-type DNA helicase gene often result in chromosomal instability, causing high frequency of carcinogenesis and progeria. These genes have been found to be expressed in various human tissues. Therefore, it can be said that the human RecQ-type gene is one of the genes encoding the DNA helicase that is involved in maintaining the basic homeostasis of the living body.

 On the other hand, RecQ5 / 3 of the present invention is a RecQ5-type DNA helicase having a localization activity in the nucleus where the DM helicase should exhibit its original function. Based on these facts, elucidation of the expression control of human RecQ5) 3 is useful for elucidating the mechanism that controls the homeostasis of living organisms, and is a novel drug for maintaining basic homeostasis of living organisms. It is useful not only for the creation of aging, but also for research to elucidate the relationship with aging.

 The various reagents according to the present invention for detecting the RecQ53 protein and the gene encoding the RecQ53 protein are useful for detecting and diagnosing a disease caused by the gene encoding a protein having a helicinase activity. is there.

The present invention relates to a polynucleotide comprising a nucleotide sequence corresponding to 1390 to 3703 in the nucleotide sequence of SEQ ID NO: 1, or a DNA comprising at least a nucleotide sequence complementary to a complementary strand thereof. In the present invention, complementary is a target Not only when the nucleotide sequence is completely complementary to the nucleotide sequence, but the nucleotide sequence of the DNA is at least 80, preferably 90% or more, and more preferably 95% or more with the completely complementary nucleotide sequence. Including the case. DNA that contains a base sequence complementary to the DNA having the target base sequence and that can hybridize to DNA consisting of the target base sequence under given stringency is used for detection of the target base sequence. It is useful as a probe or a primer for synthesis.

 Hybridization is performed using the DNA according to the present invention as an oligonucleotide probe, and RNA or DNA can be detected by Southern or Northern blotting. In addition, examples of the oligonucleotide probe include a DNA probe and an RNA probe. In designing an oligonucleotide, an arbitrary region of the region corresponding to 1390 to 3703 of the cDNA encoding RecQ53 is selected. In particular, an open reading frame region is preferable. In order to set the nucleotide sequence of the probe specific to RecQ5 i3, not only the nucleotide sequence selected from the nucleotide sequences corresponding to 1390 to 3703 in SEQ ID NO: 1 but also the portion continuous to 1390 to 3703 You can also select an area. Even for a probe that recognizes a region including 1390 to 3703, a RecQ5i3-specific nucleotide sequence can be selected. The length of the oligonucleotide used as the probe is not particularly limited as long as it is at least 15 bases and can specifically hybridize with the target base sequence under stringent conditions.

For gene detection, a gene amplification method such as a PCR method can also be used. The primer required for PCR can be set based on the nucleotide sequence described in Nucleotide Sequence: 1. The segment to be amplified in PCR is set so as to include a nucleotide sequence corresponding to 1390 to 3703 in the nucleotide sequence shown in SEQ ID NO: 1, similarly to the target nucleotide sequence of the probe. DNA encoding a region corresponding to about 580 amino acid residues on the C-terminal side, which is not found in RecQ5a but is specifically found in RecQ5 / 3 of the present invention, is important as an amplified segment of PCR. Specifically, a combination encoding a region encoding this region as a reverse-side primer and selecting a forward-side primer from a region common to RecQa. This allows specific amplification of the RecQ53 gene of the present invention. The length used for PCR is not particularly limited as long as it can specifically hybridize with the target base sequence under stringent conditions. Generally, an oligonucleotide having a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp is used for the primer.

 The hybridization assay PCR can be applied to any sample that may contain DNA or RNA, such as human tissues and blood cells. Methods for extracting DNA and RNA from these samples are known. When amplifying RNA such as mRNA, use RT-PCR, which synthesizes cDNA in advance using reverse transcriptase and uses this as a type II for PCR. Alternatively, hybridization assay PCR (including RT-PCR) can be performed in a fixed tissue (in situ).

 When detecting the presence of a target base sequence by hybridization assay or PCR, many methods for labeling a probe or primer and obtaining a signal by various detection methods are known. Any of these known access formats can be applied to the present invention.

 Further, the antibody against RecQ5i3 or RecQ53 homolog of the present invention is useful for analyzing the expression level at the protein level and detecting a mutation. For example, the expression level of RecQ5 i3 in a tissue can be determined by Western blotting. Alternatively, intracellular localization of RecQ5β can be revealed by immunochemical staining methods. Since the nuclear localization activity of the RecQ5 i3 or a homolog thereof of the present invention is an important feature, a reagent capable of clarifying intracellular localization is of great importance. Furthermore, the protein of the present invention contained in a body fluid can be quantified by a known immunoassay method such as ELISA or RIA.

Further, the present invention relates to a transgenic animal into which a gene modified to increase or decrease the expression level of RecQ53 or a homolog gene thereof has been introduced. The transgenic animal according to the present invention is characterized by its phenotypic analysis, Gives information necessary for performance analysis. In addition, knockout animals deficient in the function of RecQ53 or its homolog gene are useful as model animals for chromosomal instability diseases due to mutations in RecQ53) 3. By introducing mutations such as deletions, substitutions, additions, and insertions into some of the important sites that regulate gene expression on the genome, the expression level of the RecQ5 (or its homolog) gene can be increased. Can be modified to rise or fall artificially compared to the original level. Sites that affect gene expression include enhancers, promoters, and introns. As the vector containing the gene into which the mutation has been introduced, there are two types of vectors, that is, a vector that overexpresses the RecQ5-type gene of each animal species and an antisense vector that suppresses the expression. Examples of these vectors include pcDNA, pRC / RSV (Invitrogen) and the like. In each case, a drug for positive selection for gene selection (eg, neomycin) is linked to the resistance gene.

 Based on the present invention, antisense DNA can be designed for the DNA of RecQ53 or RecQ53) 3 homolog or a part thereof. An antisense DNA comprising an antisense sequence of at least 15 bp and targeting the hairpin loop region predicted on the mRNA can be expected to have an antisense effect.

 Methods for introducing genes into cells are known. For example, a microinjection method, ie, electroporation, in which vector DNA is directly injected into cells, can be used. Fertilized eggs and ES cells are used as the cells used to introduce the vector DNA. Among them, ES cells have the advantage of being able to be cultured outside the body and being able to generate complete individuals from these cells. Therefore, a method using various ES cells is more efficient and preferable. Examples of ES cells include TT2 cells (Shinichi Aizawa, Gene Evening Getter, 1995, Yodosha).

The vector DNA containing the mouse RecQ5 gene is introduced into ES cells by electroporation, and positively selected with neomycin to obtain the desired mutant ES cells. The ES cells described above are injected into a blastocyst or an 8-cell stage embryo using a capillary tube or the like. Then the embryo Either transfer the blastocyst or 8-cell stage embryo directly into the fetal oviduct, or transfer the blastocyst that has grown to blastocyst after culturing for one day and transfer it to the foster parent. Select chimeric animals from offspring. An animal having a high chimeric contribution rate is highly likely to be a germ line, but by crossing the chimeric animal with a normal animal, it can be confirmed that the animal is a germ line chimeric animal. By mating a germ-line chimeric animal with a normal animal, a heterozygous animal can be obtained. By mating heterozygotes, a homozygous animal can be obtained.

 According to the present invention, knockout animals in which the function of the RecQ53 (or a homolog thereof) gene has been lost can also be produced. The creation of a knockout mouse is described below using a mouse as an example. First, genomic DNA containing the mouse RecQ5 gene is obtained, and a vector is constructed in which the neo resistance gene is inserted into one of the exons. Genomic DNA can be obtained by PCR from genomic DNA prepared from mouse ES cells or from genomic libraries. This operation destroys the function of this exon. At the same time, a thymidine kinase (tk) or diphtheria (DT) toxin gene for negative selection is linked to this vector. The vector DNA is introduced into ES cells by electoporation. Next, the cells are cultured in the presence of neomycin for positive selection and FIAU (fluoro iodoadenosyl urac i 1), a nucleic acid analog for negative selection, or diphtheria toxin. By this operation, diphtheria toxin-sensitive cells that have undergone non-homologous recombination and G418-sensitive cells that do not undergo any recombination are removed, and only cells that have undergone homologous recombination survive. In cells that have undergone this homologous recombination, the gene containing the disrupted exon is knocked out. The resulting cells are injected into mouse blastocysts or 8-cell stage embryos. Thereafter, a knockout mouse can be produced by the same method as that for producing a transgenic animal.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram schematically showing the genomic structure of the human RecQ5 gene. Three RecQ5 The nucleotide sequence number of the cDNA corresponding to the exon common to isoforms is in bold, the nucleotide sequence number of the cDNA specific to RecQ53 is in thin font, and the nucleotide sequence number of the cDNA specific to RecQ53 is in italics. The base sequence number of the cDNA specific to RecQ5 was shown as 0. FIG. 2 is a diagram schematically showing the proper use of exons in the RecQ5 isoform. FIG. 3 is a diagram schematically showing the structure of the RecQ5 isoform.

 FIG. 4 is a photograph showing the results of Northern blot analysis in which the expression of RecQ53 mRNA in human organs was analyzed. RecQ5 β-specific cDNA was used as a probe.

 Two

a; heart; b; brain; c; placenta; d; lungs; e; liver 5 spleen; f; skeletal muscle; g; kidneys; h: spleen; i; spleen; j; thymus; k; m: ovary, n: small intestine, 0: large intestine, p: peripheral blood lymphocyte

 FIG. 5 is a schematic diagram showing the structure of the HA epitope-human RecQ5 isoform fusion protein expression vector, and a photograph showing the analysis result of the expression state of the RecQ5 isoform protein in human 293EBNA cells.

a: Structure of expression vector: An expression vector of 246 amino acids at the C-terminal of RecQ5, RecQ5 / 3, RecQ5r and RecQ5β was constructed based on the pcDNA3 plasmid. The N-terminus of each construct has an influenza hemagglutinin epitope (HA).

b: Photo showing the results of expression analysis by Western blot: pcDNA3 plasmid as a mock and four types of RecQ5 expression vectors were expressed in 293EBNA cells, and the cell extract was fractionated by SDS-PAGE and anti-HA antibody was used. Detected.

 FIG. 6 is a photograph showing the result of analyzing the localization of RecQ5 isoform protein in human 293EBNA cells.

Panels a to h (green) show the localization of RecQ5 isoform protein detected with anti-HA antibody. The right side (blue) of the panel shows the nucleus stained with DAPI.

 FIG. 7 is a photograph showing the analysis result of the interaction between RecQ53 and topoisomerase 3α and 3 by Western blotting.

RecQ53 expression plasmid alone or with F lag It was expressed in human 293EBNA cells together with an expression plasmid for type I topoisomerase (Topoisomerasel, Topoisomerase3, Topoisomerase 3/3). RecQ53 co-precipitates with Toboi somerase 3 α and / 3.

a: Expression of RecQ53 in each expression cell was detected with an anti-HA antibody.

b: Expression of type I topoisomerase in each expression cell was detected with an anti-Flag antibody. c: The extract of each expression cell was immunoprecipitated with an anti-Flag antibody, and the precipitated protein was detected with an anti-HA antibody.

 FIG. 8 is a photograph showing the result of immunochemical detection performed to confirm the coincidence of the localization between RecQ53 and topoisomerase 3α and 3.

 The expression plasmid for RecQ5i3 having the peptide epitope was expressed in human 293ΕΒΝΑ cells together with the expression plasmids for topoisomerase 3α and | 3 having the Flag epitope, and anti-localization was performed.

Detection was performed by immunofluorescence using HA (RecQ53Z red) and anti-Flag (topoisomerase 3α or 3Z green).

a and b show the localization of RecQ5 / 3 and topoisomerase 3α,

D indicates the localization of RecQ53 and topoisomerase 3 i3.

Panels a to d (green) show the localization of topoisomerase 3α or / 3, the center of each panel (red) shows the localization of RecQ53, and the right of each panel shows the localization of each. It is a combination. The yellow part in the right panel indicates the coincidence of the localization between RecQ53 and topoisomerase 3α or j3. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

 [Example 1] Separation of RecQ5 isoform cDNA

 (Library 1 · Making filters)

Human testis-derived cDNA library constructed based on the PAP3-neo vector (Takara Shu 1 x 10 ⁶ clones were cultured on a nitrocellulose filter (HATF08250, Millipore) at 37 ° C for about 10 hours on a LB agar plate (including 50 Aig / ml a icillin). did. Escherichia coli colonies on the filter were screened with nylon filler Yuichi (Biodyne A,

Pall) and the original nitrocellulose filter and replica Nylon filter were cultured at 37 ° C for 3 hours on an LB agar plate. Store the original nitrocellulose fil at 4 ° C, and use a replica nylon filter at 37 ° C with LB agar monoflate (including 50 ng / ml ampici 11 in, 170 g / ml chloramphenicol) ± The culture was performed for 12 hours. Denature the nylon filter (0.5 N N'aOH, 1.5 M

XaCl) for 5 minutes, neutralized with a neutralizing solution (0.5 M Tris-HCl (H7.4), 1.5 M NaCl), immersed in 2xSSC, air-dried, and heat-treated at 80 ° C for 2 hours. These filters were shaken at 50 ° C for 20 minutes in a pre-wash solution (3 X SSC, 0.5 SDS, 1 mM EDTA) and air-dried.

(First screening)

Human RecQ5 cDNA fragment (. 1.3 kb, S. Kitao et al, 1998, Genomics, 54, pp443- 452) a Random Primer DNA Labeling Kit Ver.2 [shed - ³² P] - using (Takara Shuzo) labeled with dCTP The obtained sample was used as a probe. Library pre-hybridized in a hybridization solution (5 X SS 10x Denhardt's) at 60 ° C for 3 hours — 'in a hybridization solution mixed with a filter and a probe And hybridized at 65 ° C for 20 hours. 0.5 SSC / 0.1¾ SDS solution at 65 ° C, 15 minutes, 0.5

X SSC / 0. \% SDS solution at 65 ° C, 30 minutes, 0.1 x SSC / 0.1¾ SDS solution at 65 ° C, 30 minutes, and 70 ° C in 0.1 X SSC / 0.1% SDS solution C, washed for 30 minutes and exposed to X-ray film (Kodak).

 (Second screening)

The original colony of Nitrocellulose 'fil corresponding to the positive signal on the developed X-ray film was suspended in the LB medium, and the nitrocellulose was adhered to the LB agar plate (containing 50 g / ml immediately icillin). The cells were cultured on a cellulose filter at 37 ° C for about 10 hours. Transfer the E. coli colonies on the filter to a nylon filter, The original and replica filters were cultured on LB agar plates for 373 hours. Store the original nitrocellulose fill at 1 ° C at 4 ° C. Replica nylon fill at 37 ° on a LB agar plate (including 50 g / ml a immediately icillin, 170 g / ml chloramphenicol). Incubate for 12 hours.

(0.5 X NaOH, 1.5 M aCl) for 5 minutes, neutralize with neutralizing solution (0.5 M Tris-Cl (pH 7.4), 1.5 M NaCl), immerse in 1 x SSC, air-dry, and C, heat treated for 2 hours. The filters prewashing solution (3 xSSC, 0.5¾ SDS, 1 mM EDTA) 50 ° in (:., Shaken for 2 0 minutes, and air dried in the same manner as first screening a human RecQ5 cDNA fragment [- ² P] - Hybridization was performed using a probe labeled with dCTP as a probe.

(Determination of base sequence)

 Independent E. coli colonies were selected from the obtained positive signals, cultured in 3 ml of LB medium (including 100 gla icillin), plasmid DXA was prepared by the alkali-SDS method, and a DNA solution of 1001 was obtained. Was. Using the plasmid DNA as type I and the primers of the T7 and T3 sequences upstream and downstream of the pAP3-neo vector closing site, the 5 ′ and 3 ′ nucleotide sequences of the obtained clone were determined. Cycle sequencing by PCR was used for nucleotide sequencing. That is, PCR was performed in a reaction system to which an unlabeled primer, four kinds of fluorescently labeled nucleotides 5′-triphosphate and Taq polymerase were added. The composition of the reaction mixture is as follows.

Dye Terminator Cycle Sequencing

Ready Reaction (Applied Biosystems) 8.0 1

铸 type DNA 2.0 \

Primer (3.2 pmol / u 1) 1.0 l

 DMS0 1.0, ΐ

dH ₂ 0 7.0 Ai l

 20 1

PCR was performed at 96 ° C for 30 seconds (denaturation), at 55 ° C for 15 seconds (annealing), and at 60 for 4 minutes (extension). The reaction was performed for 25 cycles, with the reaction of the long) as one cycle. In this reaction, a DNA fragment containing a fluorescent dye is synthesized at random, and by analyzing it with a sequencer, a continuous base sequence can be finally determined. Analysis of the PCR reaction was performed with an automated DNA sequencer (model ABI 373) manufactured by Applied Biosystem. Five independent clones were isolated by the above screening operation. Soshiteko these 5 'and 3' 5 clones obtained from the nucleotide sequence of the nearly full-length containing the entire open Li one Dinh Gu frame (0RF) RecQ5a, / 3 and r cD ^T A, respectively 1 One clone was obtained for each of the clones and the partial sequence cDNA of RecQ5 / 3 and RecQ5 / 3. The nucleotide sequences of the primers used to determine the nucleotide sequences of the full-length RecQ5a, 3 and α cDNAs are as follows. The sequence number is shown in 0 following the sequence name.

 (RecQ5)

 T7 (7): 5 '-TAATACGACTCACTATAGGG-3'

T3 (8): 5 '-TTAAATTAACCCTCACTAAAGG-3'

601 (9): 5 '-CATTGTACTCTCTCCTCTCATTGC-3'

 604 (10): 5 '-ATGACTTTCGTCCTGACTACTTGC-3'

 605 (11): 5 '-AGAAGTCCTTCAGGTTCCCATAGG-3'

6A (12): 5'-ACGACTGGATGGAGGAGAAGGTCC-3 '

 6B (13): 5 '-TCCTGGTGCCGTCTCTATTACTCC-3'

 6C (14): 5 '-AAAACTCCTCCTGGGTGTCTCTCC-3'

 6F (15): 0 '-CATCTCATGGAATGATCCTGTCGG-3'

 61 (16): 5 '-GTCATTCCTGGAGTAATAGAGACG-3'

 6M (17): 5 '-CAAGACCACGCCCAGCAAAAGTGG-3'

 6N (18): 5'-ATCCCCCATGTCCAATGTGCCTGG-3 '

 6P (19): 5 '-TAGAGAAGAGAGGAAAACCGCTGG-3'

 6Q (20): 5 '-AGGCTCTGGAATGCTCTCTGGAGG-3'

6W (21): 5 '-CTTACTCGTACCTTTAGGGTTAGC-3' O sz / AV z 3d1

,,) a¾)) w9i-lssvlvi) lv3) vliv: l-

, () 8 SIsl] v] l] VLLWVii¾vvv]) V-

 , () ¾L] 09] 0]]. L]] l]] i3vLV961v ::-S0a9oi]] iLL] vlv) 3JI] VJ] v3] l--,) (:} 1ΰΰ]]) ΰνν3ν33ν1ν]] 1.ν11192:-.

 )) S3] v3] vV93¾3VJV3iv1vv: ---

 , S () D] iLSs¾]) iSSI] l) v, Vll ::-l

 0 (3) l] 93] 3vf3vlvvl! V331] ivv: ---

 ,) (] ¾1] zs 3] 1ν] ΰ1] νϋν ¥ νz I—

 s (§3] S] 33S.LV] 3VV3V] V ----§s0i] 3] v] ¾vv3] v9] vv}: .--.

 ) (3)]] 3]] ll) L] i]: s92li: vlL: -—

 ] E] 31]] i3]]] «-l

 ^) 3ν3ΰ1] 31]) 11¾¾1]] 1ί} 1 ―-, s (s)] ils] v]) ∞si] lviv ¥) 9v:-.

 S (6S3) 33 ¾ 333]: SV3VVV3VVVV --- O O))) 33) gi)) vvvv)-5 ()) n)]) 3v 9 Π s) 3iEi) v3]:-(3iw3VJ)) v) i3ii i3.l3.

 -(vs3v) vw33E] gv ss s]]: ----

 ,-§) l) isvis] dimsisl] ul3.-l.

 ) (h) J]) ss¾l) 3vlvll]) vsv] 139:-.

 (§U3

33 O1) V. 601 (9): 5 '-CATTGTACTCTCTCCTCTCATTGC-3'

 604 (10): 5 '-ATGACTTTCGTCCTGACTACTTGC-3'

 605 (11): 5 '-AGAAGTCCTTCAGGTTCCCATAGG-3'

 6A (12): 5'-ACGACTGGATGGAGGAGAAGGTCC-3 '

 6B (13): 5 '-TCCTGGTGCCGTCTCTATTACTCC-3'

 61 (16): 5 '-GTCATTCCTGGAGTAATAGAGACG-3'

 [Example 2] Determination of genomic structure of human RecQ5 gene

 (Isolation of P1 phage clone of human genomic DNA containing RecQ5 gene)

 PI D'A clones, # 15033 and # 21570, were isolated from Genome Systems Inc. by a screening method using PCR. Pl # 15033 adds primers 611 and 612 and Pl # 2

1570 was screened using primers 613 and 614, respectively. Each primer

The base sequence of — is as follows. The sequence number was shown in 0 following the sequence name.

611 (36): 5'-GAGCAGCCTTGTGTTTAGACCTGG-3 '

 612 (37): 5 '-ATCCCCCATGTCCAATGTGTCTGG-3'

 613 (38): 5'-GATATAAGATTGCGTGGGTTCTGC-3 '

 614 (39): 5 '-CGTGGTCCGCCCAAGAATTAAAGG-3'

 (Preparation of PI DM)

 Escherichia coli harboring the P1 clone containing the human RecQ5 gene was cultured in 120 ml of LB medium (containing 100 ug / ml immediately icillin) for 12 hours, and then added with 0.5 mM IPTG for another 5 hours. Plasmid DNA was prepared by the alkali-SDS method to obtain an 800 zl DNA solution. In order to determine the exon Z intron boundary sequence of the human RecQ5 gene, the above-mentioned two types of P1 DNA were used as a type III and the nucleotide sequence was determined using a primer corresponding to the RecQ5 cDNA sequence. For sequencing, the cycle sequencing method was used as described above. The composition of the reaction mixture is as follows.

Dye Terminator Cycle Sequencing

Ready Reaction (Applied Biosysterns) 8.0 n 1 Type II DNA 8.0 1

Primer (3.2 pmol / z 1) 1.0 n 1

DMSO 1.0 111

dH, 0 2.0 1

Total 20 a 1

 The nucleotide sequences of the primers used for the nucleotide sequence determination are as follows. The sequence number is shown in parentheses following the sequence name.

601 (9): 5 '-CATTGTACTCTCTCCTCTCATTGC-3'

604 (10): 5 '-ATGACTTTCGTCCTGACTACTTGC-3'

6Α (12): 5 '-ACGACTGGATGGAGGAGAAGGTCC-3'

6B (13): 5 '-TCCTGGTGCCGTCTCTATTACTCC-3'

61 (16): 5 '-GTCATTCCTGGAGTAATAGAGACG-3'

6L (40): 5 '-TTATCAGCCTCCTGTCCAAGAGCC-3'

6S (41): 5 '-TCTGTGTTCTTGGCTAAGATGAGC-3'

501 (55): 5 '-AGCTGGAGCAAGACCTGCATCG-3'

502 (23): 5 '-CATGAGGACCGCTCTGAGAAGG-3'

512 (32): 5 '-GCGATGCTCTGAGAATCCTTGTGG-3'

513 (33): 5 '-AGGAGCCTTTGGGGAAACCAGC-3'

514 (34): 5 '-CGTGTTGACTGGCGGTTGCTGC-3'

5G01 (34): 5 '-GGTTCTGGCAGTGGTCGCAGC-3'

5G02 (43): 5 '-CTTCTATCTTGGGGTCTTTGC-3'

5G03 (44): 5 '-GCCAGTCAACACGTACCGCTG-3'

5G04 (45): 5 '-GTGTCCCTTGGTAGCCTACGG-3'

5G06 (46): 5 '-AGGCAAAAGACCAATAGAGAAGTG-3'

5G07 (47): 5 '-ACTAGGCAATCCACCCTGAGC-3'

5G09 (48): 5 '-CACAAGCGGGAGTGGAACCTC-3' Dimension

 : 5 '-GGTGCCTGCCCCTCCTGTGAG-3'

 5G15 (50): 5 '-CCTGTAGACCACCCTCCCAAG-3'

 5G16 (51): 5'-TGGCTGCGGCATTGTGTACTG-3 '

 5G17 (52): 5 '-AGCACCTAAGTTGTAGCCATG-3

 : 5 '-CCACCTGATACCTTGTGCCTG-3'

 5G19 (54): 5 '-CCGTCGTAGCTGAGGTCAAGG-3'

 5G20 (55): 5 '-TAGAAAGGGGTGAGGCACTTG-3'

 5G21 (56): 5 '-TGCAATAAGATGGTGGGTCAC-3'

 Comparing the obtained nucleotide sequence with the nucleotide sequences of RecQ5.3 and cDXA;

 Thetronic border sequence was determined. The results are shown in Table 1. Figure 1 shows the exon Z intron structure of the human RecQ5 gene. From these results, it is clear that the human RecQ5 gene is composed of 19 exons and 18 introns, and that RecQ5a, / 3 and isoforms are generated by using these exons properly by splicing. became. Fig. 2 shows the use of exons for RecQ5a, / 3 and isoform, and Fig. 3 shows the structure of the proteins encoded by them.

Table 1. Exon Z intron boundary sequences for RecQ5a, 3 and A

^

 S ν03} ν ::

 c \ i co CD record N oo σ> o CVJ C in co οο σ

[Example 3] Human RecQ5 gene 3 isoform mRNA expression analysis

(Preparation of probe)

By digesting the 6HA-RecQ53 plasmid (described below) with the restriction enzymes EcoRV and Xhol, An about 1.7 kb cDXA fragment specific for RecQ5 / 3 was prepared, and 30 ng of this cD'A fragment was labeled with [a- ³² P] dCTP (ΈΝ ·). Labeling was performed using Random Primer DNA Labeling Kit Ver.2 (Takara Shuzo) according to the attached protocol. A Multiple Tissue Northern Blot membrane (Clontech) in which 2 / g of poly A ⁺ RNA derived from various human tissues previously treated with a 5 × SSPE solution was blotted was mixed with 10 ml of a hybridization solution (5 × SSPE, They were immersed in 10 x Denhardt's, 50¾ formamide, ISDS, 100 / ^ g / ml salmon sperm DNA) and prehybridized at 42 ° C for 2 hours. Then, hybridization was carried out at 42 ° C for 20 hours in a new 5 ml hybridization solution containing Rec Q5) 3 specific probe labeled with [a- ³² P] dCTP. 2 X SSC / 0. \% SDS solution at room temperature, 15 minutes, 2 X SSC / 0. \% SDS solution at 65 ° C, 30 minutes, 0.2 x SSC / 0. \% SDS solution After washing at 65 ° C. for 15 minutes and further in a 0.2 × SSC / 0.1% SDS solution at 65 ° (:, 15 minutes), analysis was carried out using a BAS1500 system (Fuji II).

 As a result, the mRNA of the human RecQ5 gene (3 isoforms was expressed in all of the 16 types of tissues examined, and particularly remarkably high expression was observed in the testis (Fig. 4).

 [Example 4] Construction of expression vector of human RecQ5 isoform in mammalian cells (Construction of expression vector having influenza hemagglutinin (HA) epitope) Mammalian cell expression vector pcDN'A3 (Invitrogen) Synthetic oligo DNA (5′-GCGGCCGCCACCATGGCCTAC CCATACGATGTTCCGGATTACGCTAGCCTCCTCGAG-3′Z SEQ ID NO: 57) encoding a HA epitope having a translation initiation ATG codon at the Notl-Xhol site of E. coli was incorporated. The synthetic oligo DNA (5'-GCTAGTCTCTACCCATACGATGTTCCGGATTACGCTAGC-3'Z SEQ ID NO: 58), which encodes the HA epitope, is repeatedly inserted into the. \ Hel site on the 3 'side of the HA epitope. An expression plasmid PCDNA-6HA encoding an amino acid sequence (M number: 67) in which HA peptide was repeated 6 times was constructed.

 (Modification near the 5 'end of the ATG codon for translation initiation of the RecQ5 isoform)

RecQ5, / 3, and 0 containing the full-length 0RF cloned in the pAP3-neo vector Seven cDNAs were prepared by digestion with the restriction enzymes EcoRI and Xotl and subcloned into the EcoRI-'otl site of plasmid Blue Yuichi pBluescriptll KS +. Translation start common to three isoforms A sense primer with two restriction enzyme sites in the order of 5'-EcoRI-heI-3 'adjacent to the 5' side of the ATG codon (503: 5'-ATAGAATTCGCTAGCATGAGCAG CCACCATACCACCTTTCC -3 '/' SEQ ID NO: 59) and an antisense primer downstream of 'col' at position 273 commonly present in the three isoforms (Q6V: 5'-GTCCACTTGGTCCTGA GTCAAAGC-3 'Z SEQ ID NO: 60) Was used to perform PCR. A common cDNA fragment obtained by digesting the amplified DNA fragment with restriction enzymes EcoRI and Ncol was replaced with EcoR-Ncol portions of three isoforms subcloned into a plasmid vector pBluescriptll KS +. By the above operation, all 0RFs of these three isoforms can be cut out with restriction enzymes Nhel and Xhol.

 (Construction of RecQ5 isoform expression vector with HA epitope)

 cDN'A of three isoforms subcloned into pBluescriptll KS + and modified on the 5 'side of the ATG codon for translation initiation are cut out with heI and Xh01, and the Nhel-Xhol site of pcD \ A-6HA described above. Were cloned to construct 6HA-RecQ5, 6HA-RecQ53 and 6HA-RecQ5r (FIG. 5a). By these expression vectors, a protein in which the HA epitope repeated six times is fused to the N-terminal side of the RecQ5 isoform in an in-frame is expressed in mammalian cells and can be detected by anti-HA antibody. .

 Also, since the 3 isoforms have an endogenous Nhel sequence at position 2396, 6HA-RecQ5; 3 is completely digested with the restriction enzyme Nhel, and ligated, so that the translation start ATG codon is adjacent to the 5 'side of the ATG codon. 6HA-RecQ53c can be constructed in which approximately 2.4 kb between the existing Nhel site and the endogenous Nhel site has been removed (Figure 5a). From this expression vector, the C-terminal 246 amino acids (746-991) of RecQ53, in which the HA peptide repeated six times on the N-terminal side is fused in frame, are expressed.

[Example 5] Expression of HA epitope-human RecQ5 isoform fusion protein in mammalian cells (Gene transfer)

 Human fetal kidney-derived 293EBNA cells (Invitrogen) were cultured at 37 ° C in Doulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. Under confluent conditions, the cells were washed with phosphate buffered saline (PBS), treated with 0.25% trypsin solution, detached from the culture dishes, and subcultured at 4-fold dilution. RecQ5 isoform expression vector and control vector PCDXA-6HA (described above) 5 ^ g plasmid DNA 0.2 ml 0 PTI-MEM culture solution (mixed with Gibco BRIJ and ribofectamine 18 1 The mixture mixed with 2 ml of 0PTI-MEM culture solution was further mixed and allowed to stand at room temperature for 45 minutes 60-70% confluent 293ΕΒΆ cells were cultured in 2.5 ml of 0PTI-MEM culture solution in the culture dish described above. And added 1.6 ml of 0PTI-MEM culture solution to the DNA-ribofectamine solution left to stand for 45 minutes, and incubated at 37 ° C for 5 hours. A DMEM culture solution containing fetal serum was added, and the cells were further cultured at 37C for 19 hours, replaced with 5 ml of a DMEM culture solution containing 10% fetal bovine serum, and further cultured for 24 hours.

 (Preparation of cell extract)

 The transfected cells are collected by trypsin solution treatment, washed with PBS, and then eluted with 300 1 [50 mM Tris-Cl (pH 8.0), 150 mM aCl, 0.5 mM N'P-40. PMS F]. After standing on ice for 30 minutes, the mixture was centrifuged at 15000 rpm for 30 minutes with a micro high-speed centrifuge, and the supernatant was used as a cell extract.

 (Western blotting)

The cell extract 101 was mixed with the SDSZ sample buffer, heated at 98 ° C for 5 minutes, and then subjected to SDS-PAGE using 7.5% acrylamide gel. The protein separated in the gel was transferred to a PVDF membrane (Ibobilon, Millipore), and subjected to 5 blocking in 5% skim milk ZPBS4. PVDF membrane for 1 hour at room temperature in Egret anti-influenza hemagglutinin (HA) antibody (Santa Cruz Biotechnology, Inc.) diluted to 2.0 g / inl with PBS containing 0.05% Tween 20 as primary antibody (PBS-T) Was shaken gently and washed four times with PBS-T. 2000% with 5% skim milk ZPBS as secondary antibody The PVDF membrane was gently shaken for 1 hour at room temperature in an anti-Peagle-Emmoglobulin antibody (Amersham) labeled with horseradish peroxidase (HRP) diluted 1-fold, and washed four times with PBS-T. After immersing the PVDF membrane in PBS, detection was carried out using E (Amersham) system.

 As a result, all proteins were larger than expected size (RecQ5a: 410 aa / 46 kD, RecQ 5/3: 991 aa / 110 kD, RecQ5 r: 435 aa / 49 kD, RecQ5i3 c: 246 aa / 27 kD) A band was detected at a large position (Fig. 5b). This difference is probably due to the six-repeat HA epitope (68 aa / 8 kD) fused to each N-terminal.

 [Example 6] Analysis of intracellular localization of HA epi 1 ^ 1 punohuman RecQ5 isoform fusion protein by immunofluorescence

In the same manner as described above, the RecQ5 isoform, its derivative, and four types of expression plasmids were each introduced into 293EBNA cells and expressed. 48 hours after the gene transfer, the 293 EBNA cells were washed with PBS, detached by tribcine treatment, and suspended in 5 ml of a DMEM culture solution containing 10% fetal bovine serum. After 5 × 10 ⁴ cells were collected and washed with PBS, they were attached to a non-fluorescent slide glass (Matsunami, sSlll) by cytospin method. The cells were fixed with 10% formalin at room temperature for 10 minutes, washed with PBS-T, treated with PBS containing 0.1% Triton X-100 for 5 minutes, and blocked with PBS containing 3% skim milk for 1 hour at room temperature. . The cells were treated overnight at 4 ° C in Egret anti-HA antibody (described above) diluted to 2.5 / ig / ml with PBS containing 1.0% BSA as the primary antibody. After washing three times with PBS-T at room temperature for 10 minutes, the cells were treated with a 2000-fold diluted biotinylated anti-Peacock IgG (Biomeda) as a secondary antibody at room temperature for 1 hour, and then washed with PBS-T at room temperature. Washing was repeated 3 times for 10 minutes, followed by treatment in streptavidinated FITC (Pharmingen) at 5 _// 1111 for 1 hour at room temperature. After washing 3 times with PBS-T at room temperature for 10 minutes, nuclear staining was performed with a 50% dariserin solution containing 3 xg / ml DAPI (4 ', 6-diamidino-2-phenyl indole). Was. The subcellular localization of the HA fusion protein was performed using a fluorescence microscope (Nikon). The results are shown in FIG. In the figure, the left side of each panel is the localization of each RecQ5 isoform, and the right side is the DAPI staining image of the nucleus. RecQ5a and RecQ5a were localized in the cytoplasm, and RecQ53 was localized in the nucleus. The C-terminal 246 amino acids of RecQ53 (RecQ3c) were also localized in the nucleus. From the above, of the three RecQ5 isoforms, the smaller one (RecQ5a and RecQ5a) is localized in the cytoplasm, the larger one (RecQ5 / 3) is localized in the nucleus, and the C-terminal 246 amino acids of RecQ53. Was thought to have a nuclear translocation signal.

 [Example 7] Analysis of interaction between human type I topoisomerase and RecQ5i3

 (Construction of Expression Vector for Human Type I Topoisomerase in Mammalian Cells) Flag Epitope with ATG codon at the No 11 -Xho I site of mammalian cell expression vector -PCDNA3 (Invitrogen) ( A pcDNA-Flag was constructed by incorporating synthetic oligo DNA (5′-GCGGCCGCCACCATGGCCGACTACAAGGACGACGACGACAAGGCTAGCCTCCTCGAG-3′Z SEQ ID NO: 69) encoding MADYKDDDDKASZ SEQ ID NO: 68).

 CDNA containing all 0RF of topoisomerase 1 (GenBank ACCESSION J03250), topoisomerase 3a (GenBank ACCESSION U43431) and topoisomerase 3β (GenBank ACCESSION AF017146) was amplified by RT-PCR using the following primers, Cloned.

 (Topoisomerase 1)

Sense: 5'-TATTCTAGAATGAGTGGGGACCACCTCCACMCG-3 '/ SEQ ID NO: 61

Antisense: 5'-TATGTCGACGCTAMACTCATAGTCTTCATCAGCC-3 'NO SEQ ID NO: 62 (Topoisomerase 3α)

Sense: 5'-TATACTAGTATGATCTTTCCTGTCGCCCGCTACG-3'NO SEQ ID NO: 63

 5'-TATCTCGAGGCTCATCTGTTCTGAGGACAAAAGG-3 '(SEQ ID NO: 64-ase3β)

Sense: 5'-TATGCTAGCATGMGACTGTGCTCATGGTTGCTG-3 '/ SEQ ID NO: 65

Antisense: 5'-TATCTCGAGGGACAGGGTCATCATACAMGTAGG-3 '{SEQ ID NO: 66} The cDNA of HeLa cells to be type III was prepared as follows. Poly (A) ⁺ RN from HeLa cells A Reaction solution 251 containing about 1 g, dithiothreitol, dNTPs (dATP, dCTP, dGTP and dTTP), buffer for reverse transcriptase and Super Sucript II (Gibco BRL) for reverse transcriptase was used at 42T: The reaction was carried out for 30 minutes, and then RNase treatment was performed to obtain type I cDNA. The composition of the PCR reaction mixture is as follows.

铸 type DNA 10 ίΐ 1

Sense primer (20 mol / 1) 1.0 1

Antisense primer (20 pmol / / 1) 1.0 l

 10 x cDNA PCR Reaction Buffer 5.0 1

 (Clontech)

 2.5 mM d TP 4.0 1

 Advantage c thigh Polymerase Mix 1.0 1

 (Clontech)

dH ₂ 0 28 1

Total 50 1

 The PCR consists of one cycle of the reaction at 95 ° C for 2 minutes (denaturation) and 72 ° C for 3 minutes (annealing and extension), followed by 30 seconds at 94 ° C (denaturation) and 3 minutes at 70 ° C (annealing). And elongation) as one cycle, followed by 5 cycles, followed by 30 cycles at 94 ° C for 30 seconds (denaturation) and 68 ° C for 3 minutes (annealing and elongation). Was subjected to an extension reaction for 10 minutes. The ends of the amplified cDNA fragments of topoisomerase 1, topoisomerase 3α and topoisomerase 3β were digested with restriction enzymes Xbal and Sal I, Spel and X hoi, and Nhel and Xhol to obtain the pcDNA3-Flag vector. It was subcloned into the Nhel-Xhol site. In the constructed expression vector Flag-Topi. Flag-Τορ3α and “Flag-Top33, a protein in which a flag epitope is fused in-frame to the N-terminal side of these type I topoisomerases is expressed in mammalian cells, Detectable with anti-Flag antibody.

(Gene transfer into 293EBNA cells and immunoprecipitation) 6HA_RecQ5 / 3 plasmid DNAlO zg alone or mixed with 0.6 ml OPTI-MEM culture solution together with plasmid DN-AlOg of Flag-Topl, F1ag-Top3α or Flag-Top3i3, and lipofecta A mixture of Min 54 1 and 0.6 ml of 0PTI-MEM culture solution was further mixed and allowed to stand at room temperature for 45 minutes. Wash 100-70% confluent 293EBNA cells with 5 ml of 0PTI-MEM medium in a 100-band culture dish, and add 4.8 ml of OPTI-MEM medium to the DXA-ribofectamine solution, which has been left standing for 45 minutes. Was added and incubated at 37 ° C for 5 hours. Add an equal volume of DMEM culture medium containing 20% fetal calf serum, culture at 37 ° C for 19 hours, replace with 10 ml of DMEM culture solution containing 10% fetal calf serum, and culture for an additional 24 hours. Nourished.

 The transfected cells are collected by trypsin solution treatment, washed with PBS, and then 1 ml of eluate [50 mM Tris-Cl (H 8.0), 150 mM NaCl, 0.5¾ XP-40, 1 mM PMSF] Suspended in water. After standing on ice for 30 minutes, the mixture was centrifuged at 15,000 rpm for 30 minutes with a micro high-speed centrifuge, and the supernatant was used as a cell extract. 10 ii of this cell extract was mixed with the SDSZ sample'buffer and heated at 98 for 5 minutes. Add agarose beads (M2 agarose, Eastman Kodak) 201 immobilized with mouse anti-Flag monoclonal antibody (M2) to the remaining cell extract and shake gently at 4 ° C. The agarose beads were washed four times with the eluate. The agarose beads were suspended in eluate 1 containing 100 / g / ml Flag peptide, and gently shaken with 4 for 30 minutes. The mixture was centrifuged at 15,000 μm for 10 minutes in a micro high-speed centrifuge, and the immunoprecipitate eluted from the agarose beads by F1 ag peptide was mixed with SDSZ sample buffer and heated at 98 for 5 minutes.

The cell extract 10 or the immunoprecipitate was fractionated by SDS-PAGE using 7.5 acrylamide gel, and subjected to Western blotting in the same manner as described above. For detection of RecQ 5i3, a 2.0gg / ml anti-Peacock anti-HA antibody was used as a primary antibody, and an HRP-labeled anti-Peacock iminoglobulin antibody diluted 2000-fold was used as a secondary antibody. For detection of type I topoisomerase, a 5.0 / ig / ml mouse anti-Flag monoclonal antibody as the primary antibody and a 2000-fold diluted HRP-labeled anti-mouse immunoglobulin antibody as the secondary antibody (DAKO) was used.

 Fig. 7a shows the expression of RecQ53 in 293EBNA cells transfected with plasmid DNA of 6HA-RecQ5j3 plasmid DNA alone, 6HA-RecQ5iS and each combination of F1ag-Topl, Flag-Top3a or Flag-Top3i3. The expression of Topl, Τορ3α or Τορ33 in these 293EBNA cells is shown in Figure 7b. In each of the transfected 293EBXA cells, RecQ5j3 was expressed at the same level, and the expression of type I topoisomerase was specifically confirmed in each combination. The expected molecular weights of the proteins Τορ1, Τορ3α and Τορ3 / 3 are 91 kD, 14422 kD and 97 kD, respectively, which are almost the same as the size of the band detected by Western blotting using anti-Flag antibody. I was doing it.

 Extracts were prepared from 293EBNA cells transfected with the expression vector of each combination, and immunoprecipitated with anti-Flag antibody agarose.6HA-RecQ5 / 3 and Flag-Top3, or 6HA-RecQ53 and Flag -RecQ5 β protein was detected in immunoprecipitates derived from extracts of cells co-expressing -Top3 / 3. This result suggests that RecQ5 β protein interacts with topoisomerase 3α protein or topoisomerase 33 protein in the nucleus of cells.

 [Example 8] Analysis of nuclear localization of RecQ53, topoisomerase 3α and topoisomerase 33

 (Gene transfer into 293ΕΒΝΑ cells and expression)

6ΗΑ- RecQ53 plasmid DNA together with Fiag-Τορ3α or Flag-Top33 plasmid DNA mixed with 0.2 ml OPTI-MEM culture solution, and Ribofectamine 18/1 in 0.2 ml OPTI-MEM culture solution Was further mixed and allowed to stand at room temperature for 45 minutes. 60 Wash 60-70% confluent 293EBNA cells with 2.5 ml of OPTI-MEM culture medium in a bandit culture dish, and add 1.6 ml of OPTI-MEM culture solution to the D'A-lipofectamine solution left for 45 minutes. Was added and the mixture was kept at 37 ° C for 5 hours. Add an equal volume of DMEM culture solution containing 20% fetal calf serum, and incubate at 37 ° C for 19 hours. 交換 Replaced with DMEM culture solution containing fetal serum and cultured for another 24 hours.

 (Immunofluorescence)

The 293EBNA cells 48 hours after gene transfer were washed with PBS, detached by trypsin treatment, and suspended in 5 ml of DMEM culture solution containing 10% fetal bovine serum. After 5 × 10 ⁴ cells were sorted and washed with PBS, they were adsorbed to a non-fluorescent slide glass by the cytospin method. Fix with 10% formalin at room temperature for 10 minutes, wash with PBS-T, treat with PBS containing 0.1% Triton X-100 for 5 minutes, and block with PBS containing 3% skim milk for 1 hour at room temperature. went. The cells were treated with a rabbit ego anti-HA antibody diluted to 2.5 / g / ml in PBS containing 1.0% BSA as a primary antibody and a mouse anti-Flag antibody diluted to 10 ig / ml at 4 ° C. After washing three times for 10 minutes at room temperature in PBS-T, the cells were incubated in a 2000-fold diluted biotin-labeled anti-money IgG and a 40-fold diluted FITC-labeled anti-mouse IgG (Zymed) for 1 hour at room temperature. After the treatment, washing with PBS-T at room temperature for 10 minutes was repeated three times. After treatment with a 20-fold diluted avidin-labeled Texas Red (Oncor) at room temperature for 1 hour, washing with PBS-T at room temperature for 10 minutes three times, the localization of both proteins was determined by scanning laser microscopy ( Olymp us, Fluoview).

 Like the RecQ53 protein localized in the nucleus, the topoisomerase 3α protein was localized in the nucleus, and their localization in the nucleus coincided (Fig. 8a, b). In addition, the topoisomerase 3/3 protein was also localized in the nucleus, and the location in the nucleus was consistent with that of RecQ5 / 3 protein (Fig. 8c, d). Together with the above results and the results of immunoprecipitation, it was shown that RecQ53 protein interacts with topoisomerase 3a protein or topoisomerase 3] 3 protein in the nucleus. Industrial applicability

The present invention has provided a nuclear-localized RecQ5-type DNA helicase. Based on the previous findings that DNA helicases support chromosome stability, nuclear localization is an important factor that enables the analysis of DNA helicases' intrinsic functions. Assuming that, for example, 0. of the DNA that makes up the chromosome in the nucleus was used for gene expression, the concentration would be as high as 20M. Furthermore, if mRNA of several hundred times coexists, the risk of “entanglement” between single-stranded DNA or between single-stranded DNA and RNA is significantly increased in the nucleus. become. Under such circumstances, it can be sufficiently predicted that ligase, which is also called a DNA unwinding enzyme and has an action of unraveling double-stranded DNA into single-stranded DNA, plays an important role. It is a very interesting fact that the known RecQ5-type DNA helicase (RecQ5α) located in the cytoplasm lacks the C-terminal structure characteristic of RecQ5β according to the present invention.

 Furthermore, immunoprecipitation experiments have shown that RecQ5 / 3 of the present invention interacts with topoisomerases 3 and 3 of the three type I topoisomerases. In addition, immunofluorescence analysis of the cells showed localization agreement between topoisomerase 3α and) 3 and RecQ53. Considering these findings and the high expression of RecQ53 in the testis, RecQ5β of the present invention interacts with topoisomerase 3 and / 3 in the nucleus of cells, and is involved in DNA metabolic reactions such as recombination and repair. It is suggested that they are closely involved. Furthermore, RecQ5β of the present invention, which has an isoform (RecQ5) localized in the cytoplasm, is considered to be highly likely to be a causative gene of chromosomal instability disease. Therefore, Rec Q53 of the present invention is expected to be a useful protein as a diagnostic indicator of chromosomal instability disease or in the treatment.

Claims

The scope of the claims

 1. The polynucleotide according to any one of the following (a) to (d):

 (a) a polynucleotide comprising a protein coding region having the nucleotide sequence described in the sequence;

(b) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2;

(c) an amino acid sequence represented by SEQ ID NO: 2 consisting of an amino acid sequence in which one or several amino acids have been modified by deletion, addition, insertion, and substitution with Z or another amino acid, and have helicase activity. A polynucleotide encoding a nuclear localization protein having the same.

 (d) a protein that is encoded by DNA that hybridizes under stringent conditions to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and encodes a nuclear localization protein having helicase activity Polynucleotides ,.

 2. A protein encoded by the polynucleotide of claim 1.

 3. A vector into which the polynucleotide according to claim 1 has been inserted.

 4. A transformant carrying the polynucleotide of claim 1 or the vector of claim 3.

 5. The method for producing a protein according to claim 2, comprising a step of culturing the transformant according to claim 5 and collecting an expression product.

 6. A polynucleotide comprising a nucleotide sequence corresponding to 1390 to 3703 in the nucleotide sequence of SEQ ID NO: 1, or a polynucleotide having a chain length of at least 15 nucleotides, comprising a nucleotide sequence complementary to a complementary strand thereof.

 7. The primer for polynucleotide synthesis according to claim 1, consisting of the polynucleotide according to claim 6.

 8. The probe for detecting a polynucleotide according to claim 1, comprising the polynucleotide according to claim 6.

9. Antisense D against the polynucleotide or a part thereof according to claim 1. A partial peptide consisting of an amino acid sequence corresponding to positions 411 to 991 in the amino acid sequence of SEQ ID NO: 2.

An antibody that recognizes a partial peptide consisting of an amino acid sequence corresponding to positions 411-1991 in the amino acid sequence of SEQ ID NO: 2.

An immunoassay method comprising a step of contacting the antibody according to claim 11 with a protein comprising the amino acid sequence according to SEQ ID NO: 2, or a partial peptide thereof, and detecting an immunological reaction between the two.

A reagent for detecting a protein comprising the amino acid sequence of SEQ ID NO: 2 or a partial peptide thereof, comprising the antibody according to claim 11.

A transgenic non-human vertebrate having an altered expression level of the polynucleotide of claim 1.

 The transgenic non-human vertebrate according to claim 14, which is a knockout animal lacking the function of the polynucleotide according to claim 1.