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Replication-Associated Repair of Adenine:8-Oxoguanine Mispairs by MYH

2002; Elsevier BV; Volume: 12; Issue: 4 Linguagem: Inglês

10.1016/s0960-9822(02)00686-3

1879-0445

Harutoshi Hayashi, Yohei Tominaga, Seiki Hirano, Allison E. McKenna, Yusaku Nakabeppu, Yoshihiro Matsumoto,

Cancer-related Molecular Pathways

Cellular DNA is constantly exposed to the risk of oxidation. 8-oxoguanine (8-oxoG) is one of the major DNA lesions generated by oxidation, which is primarily corrected by base excision repair. When it is not repaired prior to replication, replicative DNA polymerases yield misinsertion of an adenine (A) opposite the 8-oxoG on the template strand, generating an A:8-oxoG mispair [1Shibutani S. Takeshita M. Grollman A.P. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG.Nature. 1991; 349: 431-432Crossref PubMed Scopus (1974) Google Scholar]. MYH, a mammalian homolog of Escherichia coli MutY, is a DNA glycosylase responsible for initiating base excision repair of such a mispair by excising the adenine opposite 8-oxoG [2Slupska M.M. Baikalov C. Luther W.M. Chiang J.H. Wei Y.F. Miller J.H. Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage.J. Bacteriol. 1996; 178: 3885-3892Crossref PubMed Scopus (316) Google Scholar]. Here, using an in vivo repair system, we show that DNA replication enhances the repair of the A:8-oxoG mispair. Repair efficiency was lower in MYH-deficient murine cells than in MYH-proficient cells. Transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:8-oxoG repair, indicating that a significant part of this replication-associated repair depends on MYH. Expression of a mutant MYH in which the PCNA binding motif was disrupted did not increase the repair efficiency, thus suggesting that the interaction between PCNA and MYH is critical for MYH-initiated repair of A:8-oxoG. Cellular DNA is constantly exposed to the risk of oxidation. 8-oxoguanine (8-oxoG) is one of the major DNA lesions generated by oxidation, which is primarily corrected by base excision repair. When it is not repaired prior to replication, replicative DNA polymerases yield misinsertion of an adenine (A) opposite the 8-oxoG on the template strand, generating an A:8-oxoG mispair [1Shibutani S. Takeshita M. Grollman A.P. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG.Nature. 1991; 349: 431-432Crossref PubMed Scopus (1974) Google Scholar]. MYH, a mammalian homolog of Escherichia coli MutY, is a DNA glycosylase responsible for initiating base excision repair of such a mispair by excising the adenine opposite 8-oxoG [2Slupska M.M. Baikalov C. Luther W.M. Chiang J.H. Wei Y.F. Miller J.H. Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage.J. Bacteriol. 1996; 178: 3885-3892Crossref PubMed Scopus (316) Google Scholar]. Here, using an in vivo repair system, we show that DNA replication enhances the repair of the A:8-oxoG mispair. Repair efficiency was lower in MYH-deficient murine cells than in MYH-proficient cells. Transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:8-oxoG repair, indicating that a significant part of this replication-associated repair depends on MYH. Expression of a mutant MYH in which the PCNA binding motif was disrupted did not increase the repair efficiency, thus suggesting that the interaction between PCNA and MYH is critical for MYH-initiated repair of A:8-oxoG. Human MYH protein has enzyme activities for removing an adenine at the A:8-oxoG mispair as well as a 2-hydroxyadenine (2-OH-A) at the 2-OH-A:G mispair [3Ohtsubo T. Nishioka K. Imaiso Y. Iwai S. Shimokawa H. Oda H. Fujiwara T. Nakabeppu Y. Identification of human MutY homolog (hMYH) as a repair enzyme for 2-hydroxyadenine in DNA and detection of multiple forms of hMYH located in nuclei and mitochondria.Nucleic Acids Res. 2000; 28: 1355-1364Crossref PubMed Scopus (262) Google Scholar]. A:8-oxoG mispair is formed in vivo during DNA replication by two mechanisms: either by incorporation of an adenine nucleotide opposite an 8-oxoG derived from the direct oxidation in the template strand [1Shibutani S. Takeshita M. Grollman A.P. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG.Nature. 1991; 349: 431-432Crossref PubMed Scopus (1974) Google Scholar], or by incorporation of an 8-oxodGTP that results from direct oxidation of dGTP in the nucleotide pool [4Maki H. Sekiguchi M. MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis.Nature. 1992; 355: 273-275Crossref PubMed Scopus (766) Google Scholar]. Recently, it has been demonstrated that MYH colocalizes with PCNA at DNA replication foci [5Boldogh I. Milligan D. Lee M.S. Bassett H. Lloyd R.S. McCullough A.K. hMYH cell cycle-dependent expression, subcellular localization and association with replication foci: evidence suggesting replication-coupled repair of adenine:8-oxoguanine mispairs.Nucleic Acids Res. 2001; 29: 2802-2809Crossref PubMed Scopus (127) Google Scholar]. Mammalian MYH carries a consensus motif for binding to PCNA, [QXX(I/L/M)XXFF] [6Warbrick E. The puzzle of PCNA's many partners.Bioessays. 2000; 22: 997-1006Crossref PubMed Scopus (353) Google Scholar], at its C terminus, and, indeed, it can physically interact with PCNA [7Parker A. Gu Y. Mahoney W. Lee S.H. Singh K.K. Lu A.L. Human homolog of the MutY repair protein (hMYH) physically interacts with proteins involved in long patch DNA base excision repair.J. Biol. Chem. 2001; 276: 5547-5555Crossref PubMed Scopus (185) Google Scholar]. Based on these properties of the MYH protein and the replication-dependent formation of A:8-oxoG mispairs, we proposed a hypothesis in which MYH-initiated base excision repair of A:8-oxoG should be closely associated with DNA replication through direct interaction between MYH and PCNA [8Matsumoto Y. Molecular mechanism of PCNA-dependant base excision repair.Prog. Nucleic Acid Res. Mol. Biol. 2001; 68: 129-138Crossref PubMed Google Scholar]. To test this hypothesis, we developed an in vivo repair assay system. In this system, murine cultured cells were cotransfected with two circular plasmid DNAs (Figure 1A). One plasmid carried an A:8-oxoG mispair in the lacZ α-complementation fragment, a replication cassette derived from mouse polyoma virus, and the ampicillin-resistant gene (pAmp-Py-lac) and was used as a repair substrate. The other plasmid contained the same replication cassette and the tetracycline-resistant gene (pTet-Py) and was used as a control in order to normalize the efficiencies of transfection and subsequent bacterial transformation. We also used two DNA substrates with 8-oxoG: one in which 8-oxoG was placed on the template strand for lagging strand DNA synthesis, and the other in which 8-oxoG was placed on the template strand for leading strand synthesis. After 3 days of incubation, the plasmid DNA was recovered from transfected cells and treated with E. coli MutY, exonuclease III, and mung bean nuclease to linearize the DNA substrate carrying unrepaired A:8-oxoG. The treated DNA was then used for transformation of the DH10B bacterial strain, and colonies that formed on ampicillin/IPTG/X-gal-containing plates and on tetracyclin-containing plates were counted. The DNA in which A:8-oxoG was repaired to C:G by the MYH-initiated mechanism was expected to form blue colonies, whereas the DNA in which A:8-oxoG is converted to A:T was expected to carry a stop codon in the lacZ gene and form white colonies (Figure 1B). The linearized DNA would not form colonies. When the replication-proficient DNA substrate was used for A:8-oxoG repair, the DNA that became resistant to MutY digestion was recovered with 14-fold higher efficiency than that found with the replication-deficient DNA substrate (represented by the total area of the pie charts and the numbers shown under the pie charts in Figure 2). The low yield of ampicillin-resistant colonies in the absence of replication suggests that the A:8-oxoG mispair cannot be efficiently repaired without replication (the uppermost pathway versus the lowermost pathway in Figure 1B). Accordingly, the high yield of ampicillin-resistant colonies in the replicative system is likely to result from enhancement of the repair efficiency by replication (the second and fourth pathways of Figure 1B). An alternative explanation for the difference between the replicative and nonreplicative systems is that replication did not enhance the repair efficiency, but amplified the DNA substrate following its repair. To distinguish these possibilities, we estimated the replication efficiency in our assay system by treatment with DpnI, which digests the original DNA substrates at fully methylated GATC sequences, but not the DNA that contains only hemimethylated or unmethylated GATC sequences after replication in murine cells. The percentage of replicated DNA among the total DNA recovered from murine cells was 19 ± 9% (data not shown), suggesting that the amplification of repaired DNA by replication, if any, can cause only a minor effect in this assay system. Thus, the more than 10-fold increase of A:8-oxoG repair in replication-proficient DNA substrates indicates that the repair reaction of A:8-oxoG is significantly enhanced by replication of the DNA substrate in vivo. When the DNA substrate is replicated prior to repair, the A:8-oxoG mispair will dissociate and each base will respectively serve as a template. Biochemical characterization of DNA polymerase δ reveals that this replicative enzyme mainly inserts an adenine opposite an 8-oxoG [1Shibutani S. Takeshita M. Grollman A.P. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG.Nature. 1991; 349: 431-432Crossref PubMed Scopus (1974) Google Scholar]. The progeny DNA derived from the strand containing the adenine at the mispair site is expected to form a white colony, while the progeny DNA derived from the strand containing the 8-oxoG should form a blue colony if it is repaired as expected. We sequenced 20 each of blue and white colonies from the experiments shown in Figure 2 and verified that 19 of the blue colonies (95%) contained the correctly repaired sequence (A:8-oxoG → C:G) and that 19 of the white colonies (95%) contained an A:T pair at the original mispair site. These results indicate that the replication-associated repair of 8-oxoG is relatively accurate and does not result from random nucleotide insertion opposite the 8-oxoG. We also observed that the repair efficiency of the substrate carrying 8-oxoG on the template for leading strand synthesis was higher than that of the substrate carrying 8-oxoG on the template for lagging strand synthesis, and the efficiency exceeded the expected maximum efficiency, 50% (Figure 2). There are several possible explanations provided. Firstly, this strand-specific effect may result from differences in the sequence context around the mispair or differences of the replication complexes engaged in the two strands, such as the number of loaded PCNA molecules or the involvement of DNA polymerase α. It is also possible that the preexisting A:8-oxoG mispair on the DNA substrate may be repaired before the replication fork dissociates the mispair into two strands at a replication site wherein the MYH protein is concentrated. Such prereplicative repair of an A:8-oxoG mispair may contribute in part to the replication-dependent repair in a strand-specific manner and may result in blue colony formation of greater than 50% (Figure 2, right lower panel). Another possibility is that one of the two strands of the DNA substrates may be more preferentially utilized as a template for replication, which can also provide an explanation for blue colony formation of greater than 50%. Yet another possibility is that transcription may cause strand-specific repair. However, this possibility is unlikely for two reasons: first, no known mammalian promoter exists that may cause transcription at the mispair region in the DNA substrate used; and second, no transcriptional products spanning the mispair region were detected by RT-PCR (data not shown). Nevertheless, it is difficult to rule out the possibility of an undetectable level of read-through transcription. To examine if the replication-associated repair of A:8-oxoG is indeed mediated by MYH, we compared the repair efficiencies in MYH-proficient and MYH-deficient murine cells (Y.T., S.H., and Y.N., unpublished data). In order to focus on replication-associated repair, an experiment was performed in which the DNA recovered from transfected murine cells was treated with DpnI, digesting all the unreplicated DNA. In both strand situations, the correct repair of A:8-oxoG (as assessed by the relative ratio of blue colonies to the total number of colonies) was more efficient in MYH-proficient cells than in MYH-deficient cells (Figure 3). It is not surprising that murine cells lacking MYH were able to repair A:8-oxoG at a certain level in the replication-associated manner. There are several other possible mechanisms to correct the A:8-oxoG mispair distinct from MYH-initiated base excision repair. One prominent possibility is the mechanism of replication itself. DNA polymerase δ organized in replication machinery in vivo may insert a cytosine opposite the 8-oxoG more frequently than in a purified form in vitro. Alternatively, one of the trans-lesion DNA polymerases recently identified [9Friedberg E.C. Feaver W.J. Gerlach V.L. The many faces of DNA polymerases: strategies for mutagenesis and for mutational avoidance.Proc. Natl. Acad. Sci. USA. 2000; 97: 5681-5683Crossref PubMed Scopus (222) Google Scholar] may substitute for DNA polymerase δ in bypassing the 8-oxoG on the template, incorporating a cytosine. Another possible mechanism is MSH2/MSH6-mediated long-patch mismatch repair. In Saccharomyces cerevisiae, which does not have a homolog of E. coli MutY, mismatch repair is the major pathway for repairing the A:8-oxoG mispair [10Ni T.T. Marsischky G.T. Kolodner R.D. MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxo-guanine in S. cerevisiae.Mol. Cell. 1999; 4: 439-444Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar]. Nevertheless, our data suggest that a significant fraction of replication-associated A:8-oxoG repair is mediated by MYH in mammalian cells. To further confirm the involvement of MYH in replication-associated repair of A:8-oxoG, we reintroduced the MYH protein into MYH-deficient cells by transfection with an expression vector coding for mouse MYH (Figure 4B). The expression of wild-type MYH resulted in a solid increase of repair efficiency, supporting the repair mechanism mediated by MYH. In contrast, expression of a mutant MYH wherein the consensus PCNA binding motif was disrupted did not enhance repair efficiency at all, though the expression level of mutant MYH was comparable to that of the wild-type MYH (Figure 4A). It is not clear at this moment why the strand bias of A:8-oxoG repair was not observed in MYH-transfected cells. One possible explanation is that the expression level and/or regulation of MYH from the EF-1α promoter in the transfected cells may be different from those of the endogenous MYH gene that is regulated in a cell cycle-dependent manner [5Boldogh I. Milligan D. Lee M.S. Bassett H. Lloyd R.S. McCullough A.K. hMYH cell cycle-dependent expression, subcellular localization and association with replication foci: evidence suggesting replication-coupled repair of adenine:8-oxoguanine mispairs.Nucleic Acids Res. 2001; 29: 2802-2809Crossref PubMed Scopus (127) Google Scholar] and therefore cannot reproduce the strand-biased repair. Nevertheless, the results with MYH-transfected cells suggest that the interaction between MYH and PCNA through the consensus motif is critical for replication-associated repair by MYH. MYH is one of the two mammalian DNA glycosylases that have been demonstrated to physically interact with PCNA through the consensus binding motif. The other DNA glycosylase is a nuclear form of uracil DNA glycosylase, UNG2 [11Otterlei M. Warbrick E. Nagelhus T.A. Haug T. Slupphaug G. Akbari M. Aas P.A. Steinsbekk K. Bakke O. Krokan H.E. Post-replicative base excision repair in replication foci.EMBO J. 1999; 18: 3834-3844Crossref PubMed Scopus (275) Google Scholar]. Similar to MYH, one of the targets of UNG2 is a mispair generated during replication through incorporation of a uracil opposite an adenine. UNG2 colocalizes with PCNA at replication foci in mammalian cells [11Otterlei M. Warbrick E. Nagelhus T.A. Haug T. Slupphaug G. Akbari M. Aas P.A. Steinsbekk K. Bakke O. Krokan H.E. Post-replicative base excision repair in replication foci.EMBO J. 1999; 18: 3834-3844Crossref PubMed Scopus (275) Google Scholar]. Both MYH and UNG2 also bind to RPA, which is essential for replication [12Fairman M.P. Stillman B. Cellular factors required for multiple stages of SV40 DNA replication in vitro.EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (284) Google Scholar], but not for base excision repair [13Stucki M. Pascucci B. Parlanti E. Fortini P. Wilson S.H. Hubscher U. Dogliotti E. Mammalian base excision repair by DNA polymerases delta and epsilon.Oncogene. 1998; 17: 835-843Crossref PubMed Scopus (152) Google Scholar, 14Matsumoto Y. Kim K. Hurwitz J. Gary R. Levin D.S. Tomkinson A.E. Park M.S. Reconstitution of proliferating cell nuclear antigen-dependent repair of apurinic/apyrimidinic sites with purified human proteins.J. Biol. Chem. 1999; 274: 33703-33708Crossref PubMed Scopus (138) Google Scholar]. Therefore, the interactions of MYH and UNG2 with PCNA may serve to recruit these DNA glycosylases to the replication sites, facilitating repair of two types of lesions generated by replication. In addition, 2-OH-A, which can also be repaired by MYH, is mostly derived from the insertion of 2-OH-dAMP into a newly synthesized strand, but not from direct oxidation of adenine on a template strand [3Ohtsubo T. Nishioka K. Imaiso Y. Iwai S. Shimokawa H. Oda H. Fujiwara T. Nakabeppu Y. Identification of human MutY homolog (hMYH) as a repair enzyme for 2-hydroxyadenine in DNA and detection of multiple forms of hMYH located in nuclei and mitochondria.Nucleic Acids Res. 2000; 28: 1355-1364Crossref PubMed Scopus (262) Google Scholar]; thus, its repair is also likely to depend on the replication-associated mechanism with MYH. It is not clear from currently available data whether these DNA glycosylases interact directly with the replication machinery to couple repair with replication or if they bind to PCNA molecules remaining on the replicated DNA for postreplicative repair (Figure 5). In the repair reaction with the DNA substrates used in this study, it is also possible that the preexisting A:8-oxoG is repaired before replication dissociates the mispair. However, this prereplicative repair is unlikely to work for endogenous A:8-oxoG mispairs, because the mispair formed during replication would remain for the whole cell cycle until the next round of replication, and also because PCNA, which is loaded primarily on the split side of the replication fork, would not efficiently assist MYH's function in front of the replication fork. Rather, it is more reasonable that A:8-oxoG mispairs would be repaired immediately after their formation at the replicated site. It has been proposed that PCNA could coordinate long-patch mismatch repair with replication [15Umar A. Buermeyer A.B. Simon J.A. Thomas D.C. Clark A.B. Liskay R.M. Kunkel T.A. Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis.Cell. 1996; 87: 65-73Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar]. In this scenario, the orientation of a PCNA trimer loaded on DNA may aid the mismatch repair complex in distinguishing the newly synthesized strand from the template strand. MYH may employ a similar mechanism. An adenine incorporated opposite the 8-oxoG on the template strand must be removed from the newly synthesized strand. Conversely, an A:8-oxoG mispair generated by incorporation of 8-oxodGTP opposite an adenine requires that the 8-oxoG on the newly synthesized strand must be removed, whereas the adenine on the template strand should be maintained to preserve the original genetic information. This mechanism for selective recognition of a newly synthesized strand through interaction with PCNA could prevent MYH from causing mutations by removing an adenine on the template strand. The wild-type mouse embryonic fibroblast cell line [16Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Requirement of mammalian DNA polymerase-beta in base-excision repair.Nature. 1996; 379: 183-186Crossref PubMed Scopus (754) Google Scholar] was maintained as previously described [17Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. Impairment of proliferating cell nuclear antigen-dependent apurinic/apyrimidinic site repair on linear DNA.J. Biol. Chem. 1998; 273: 898-902Crossref PubMed Scopus (173) Google Scholar]. The procedures for establishing wild-type and MYH knockout mouse embryonic stem (ES) cells will be described elsewhere (Y.T., S.H., and Y.N., unpublished data). The absence of MYH in MYH-deficient cells was confirmed by immunoblotting and a nicking assay with A:8-oxoG-containing DNA (data not shown). Before performing the repair assay, the ES cells were differentiated by omitting leukemia inhibitory factor from the medium, and they were maintained in DMEM medium supplemented with 20% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM β-mercaptoethanol. pAmp-Py-lac was constructed by inserting the polyoma replication cassette derived from pMGD20neo [18Gassmann M. Donoho G. Berg P. Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells.Proc. Natl. Acad. Sci. USA. 1995; 92: 1292-1296Crossref PubMed Scopus (64) Google Scholar, 19Camenisch G. Gruber M. Donoho G. Van Sloun P. Wenger R.H. Gassmann M. A polyoma-based episomal vector efficiently expresses exogenous genes in mouse embryonic stem cells.Nucleic Acids Res. 1996; 24: 3707-3713Crossref PubMed Scopus (19) Google Scholar] into the AflIII and SapI sites of pBS– or pBS+ (Stratagene). For the replication-deficient construct, 46 bp containing the A/T-rich region and the central palindrome of the origin core were deleted. Covalently closed circular DNA (cccDNA) that carried an A:8-oxoG mispair was prepared by essentially the same procedures as previously described [20Matsumoto Y. Base excision repair assay using Xenopus laevis oocyte extracts.Methods Mol. Biol. 1999; 113: 289-300PubMed Google Scholar]. pTet-Py was constructed by deleting the ampicillin-resistant gene from pBR322 and inserting the polyoma replication cassette into the resultant plasmid. Murine cells were seeded onto 6-well plates at a concentration of 2 × 103 cells per well approximately 24 hr before transfection. The cells in each well were cotransfected with 0.1 μg of a mispair-carrying pAmp-Py-lac and 0.5 μg pTet-Py by lipofectAMINE 2000 (Life Technologies). At 72 hr after transfection, cells were rinsed with cold phosphate-buffered saline (PBS) and then treated with 1% NP-40 in PBS for 1 min. After cells were washed with PBS, the attached nuclei were collected [21Cahill K.B. Roome A.J. Carmichael G.G. Replication-dependent transactivation of the polyomavirus late promoter.J. Virol. 1990; 64: 992-1001PubMed Google Scholar]. The plasmid DNA was recovered from the nuclei with the QIAGEN miniprep kit. The recovered DNA was incubated with 10 U MutY (Trevigen) at 37°C for 1 hr in the MutY reaction buffer (Trevigen) and subsequently incubated with exonuclease III and mung bean nuclease (both from New England Biolabs) in the buffer supplemented with 10 mM MgCl2 at 37°C for 45 min. When noted, the DNA samples were digested with 2 U DpnI (New England Biolabs). Finally, the DNA samples were used for transformation of DH10B by electroporation, and the bacteria were spread on an LB agar plate containing ampicillin, IPTG, and X-gal and on an LB agar plate containing tetracycline. The number of colonies on a tetracycline-containing plate was used for normalization of the efficiencies of murine cell transfection and bacterial transformation. A mouse MYH mutant was made by replacing two phenylalanines in its PCNA binding motif with two alanines (F500A/F501A). A wild-type or mutated MYH cDNA was inserted into the expression vector with human elongation factor 1α-subunit promoter and the C-terminal c-Myc epitope and polyhistidine tags (pEF1; Invitrogen). A total of 2 μg of the wild-type or mutant MYH expression vector or the empty vector was linearized by XmaI digestion, then used for transfection of MYH-deficient cells on 6-well plates. After 24 hr, transfected cells were split and in vivo repair assays were performed as described above. Western blot analysis was conducted with anti-c-Myc antibody (A-14, Santa Cruz Biotechnology) or anti-Actin antibody (C-11, Santa Cruz Biotechnology). We thank G. Camenisch and M. Gassmann for providing pMGD20neo plasmid, and A. Bellacosa, A. Gudzelak, M.E. Murphy, and H. Yan for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (GM57343, CA06927) and an appropriation from the Commonwealth of Pennsylvania.