Lesion Bypass Activities of Human DNA Polymerase μ
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m207297200
ISSN1083-351X
AutoresYanbin Zhang, Xiaohua Wu, Dongyu Guo, Olga Rechkoblit, John‐Stephen Taylor, Nicholas E. Geacintov, Zhi‐Gang Wang,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoDNA polymerase μ (Polμ) is a newly discovered member of the polymerase X family with unknown cellular function. The understanding of Polμ function should be facilitated by an understanding of its biochemical activities. By using purified human Polμ for biochemical analyses, we discovered the lesion bypass activities of this polymerase in response to several types of DNA damage. When it encountered a template 8-oxoguanine, abasic site, or 1,N 6-ethenoadenine, purified human Polμ efficiently bypassed the lesion. Even bulky DNA adducts such asN-2-acetylaminofluorene-adducted guanine, (+)- and (−)-trans-anti-benzo[a]pyrene-N 2-dG were unable to block the polymerase activity of human Polμ. Bypass of these simple base damage and bulky adducts was predominantly achieved by human Polμ through a deletion mechanism. The Polμ specificity of nucleotide incorporation indicates that the deletion resulted from primer realignment before translesion synthesis. Purified human Polμ also effectively bypassed a template cis-synTT dimer. However, this bypass was achieved in a mainly error-free manner with AA incorporation opposite the TT dimer. These results provide new insights into the biochemistry of human Polμ and show that efficient translesion synthesis activity is not strictly confined to the Y family polymerases. DNA polymerase μ (Polμ) is a newly discovered member of the polymerase X family with unknown cellular function. The understanding of Polμ function should be facilitated by an understanding of its biochemical activities. By using purified human Polμ for biochemical analyses, we discovered the lesion bypass activities of this polymerase in response to several types of DNA damage. When it encountered a template 8-oxoguanine, abasic site, or 1,N 6-ethenoadenine, purified human Polμ efficiently bypassed the lesion. Even bulky DNA adducts such asN-2-acetylaminofluorene-adducted guanine, (+)- and (−)-trans-anti-benzo[a]pyrene-N 2-dG were unable to block the polymerase activity of human Polμ. Bypass of these simple base damage and bulky adducts was predominantly achieved by human Polμ through a deletion mechanism. The Polμ specificity of nucleotide incorporation indicates that the deletion resulted from primer realignment before translesion synthesis. Purified human Polμ also effectively bypassed a template cis-synTT dimer. However, this bypass was achieved in a mainly error-free manner with AA incorporation opposite the TT dimer. These results provide new insights into the biochemistry of human Polμ and show that efficient translesion synthesis activity is not strictly confined to the Y family polymerases. DNA polymerase μ (Polμ) 1The abbreviations used are: Pol, DNA polymerase; BPDE, benzo[a]pyrene-trans-7,8-dihydrodiol-9, 10-epoxide; AAF, N-2-acetylaminofluorene; AP, apurinic/apyrimidinic; NHEJ, nonhomologous end joining 1The abbreviations used are: Pol, DNA polymerase; BPDE, benzo[a]pyrene-trans-7,8-dihydrodiol-9, 10-epoxide; AAF, N-2-acetylaminofluorene; AP, apurinic/apyrimidinic; NHEJ, nonhomologous end joiningis a newly discovered member of the X family polymerases (1Dominguez O. Ruiz J.F. Lain de Lera T. Garcia-Diaz M. Gonzalez M.A. Kirchhoff T. Martinez A.C. Bernad A. Blanco L. EMBO J. 2000; 19: 1731-1742Crossref PubMed Google Scholar, 2Aoufouchi S. Flatter E. Dahan A. Faili A. Bertocci B. Storck S. Delbos F. Cocea L. Gupta N. Weill J.C. Reynaud C.A. Nucleic Acids Res. 2000; 28: 3684-3693Crossref PubMed Google Scholar). Additional members in this family include Polβ, Polλ, and terminal deoxynucleotidyltransferase (1Dominguez O. Ruiz J.F. Lain de Lera T. Garcia-Diaz M. Gonzalez M.A. Kirchhoff T. Martinez A.C. Bernad A. Blanco L. EMBO J. 2000; 19: 1731-1742Crossref PubMed Google Scholar, 2Aoufouchi S. Flatter E. Dahan A. Faili A. Bertocci B. Storck S. Delbos F. Cocea L. Gupta N. Weill J.C. Reynaud C.A. Nucleic Acids Res. 2000; 28: 3684-3693Crossref PubMed Google Scholar, 3Garcia-Diaz M. Dominguez O. Lopez-Fernandez L.A. de Lera L.T. Saniger M.L. Ruiz J.F. Parraga M. Garcia-Ortiz M.J. Kirchhoff T. del Mazo J. Bernad A. Blanco L. J. Mol. Biol. 2000; 301: 851-867Crossref PubMed Scopus (246) Google Scholar). During base excision repair in higher eukaryotes, Polβ is a major repair synthesis polymerase (4Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 5Wood R.D. Shivji M.K. Carcinogenesis. 1997; 18: 605-610Crossref PubMed Scopus (144) Google Scholar, 6Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (264) Google Scholar). Terminal deoxynucleotidyltransferase catalyzes nucleotide additions to DNA in a template-independent manner (7Chang L.M. Bollum F.J. CRC Crit. Rev. Biochem. 1986; 21: 27-52Crossref PubMed Scopus (60) Google Scholar, 8Bentolila L.A. Fanton d'Andon M. Nguyen Q.T. Martinez O. Rougeon F. Doyen N. EMBO J. 1995; 14: 4221-4229Crossref PubMed Scopus (44) Google Scholar). This enzyme functions during V(D)J recombination of the immunoglobulin genes and T-cell receptor genes and is restricted to lymphoid tissues (7Chang L.M. Bollum F.J. CRC Crit. Rev. Biochem. 1986; 21: 27-52Crossref PubMed Scopus (60) Google Scholar, 8Bentolila L.A. Fanton d'Andon M. Nguyen Q.T. Martinez O. Rougeon F. Doyen N. EMBO J. 1995; 14: 4221-4229Crossref PubMed Scopus (44) Google Scholar, 9Weaver D.T. Adv. Immunol. 1995; 58: 29-85Crossref PubMed Scopus (59) Google Scholar). Cellular functions of Polλ and Polμ have not been clearly defined.Although the biochemical activities of the X family DNA polymerases appear to be quite diverse, all of the Y family DNA polymerases share a common biochemical activity: synthesis opposite DNA lesions (reviewed in Refs. 10Wang Z. Mutat. Res. 2001; 486: 59-70Crossref PubMed Scopus (85) Google Scholar, 11Wang Z. Mol. Interv. 2001; 1: 269-281PubMed Google Scholar, 12Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13Friedberg E.C. Fischhaber P.L. Kisker C. Cell. 2001; 107: 9-12Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In eukaryotes, the Y family consists of REV1 and DNA polymerases η, ι, and κ (14Ohmori H. Friedberg E.C. Fuchs R.P.P. Goodman M.F. Hanaoka F. Hinkle D. Kunkel T.A. Lawrence C.W. Livneh Z. Nohmi T. Prakash L. Prakash S. Todo T. Walker G.C. Wang Z. Woodgate R. Mol. Cell. 2001; 8: 7-8Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar). Thus, it is generally believed that a major function of the Y family DNA polymerases is to copy damaged sites of DNA during replication, a cellular process referred to as lesion bypass or translesion synthesis. Genetic studies indicate that REV1 (15Larimer F.W. Perry J.R. Hardigree A.A. J. Bacteriol. 1989; 171: 230-237Crossref PubMed Google Scholar, 16Rajpal D.K., Wu, X. Wang Z. Mutat. Res. 2000; 461: 133-143Crossref PubMed Scopus (37) Google Scholar, 17Nelson J.R. Gibbs P.E. Nowicka A.M. Hinkle D.C. Lawrence C.W. Mol. Microbiol. 2000; 37: 549-554Crossref PubMed Scopus (183) Google Scholar, 18Gibbs P.E. Wang X.D., Li, Z. McManus T.P. McGregor W.G. Lawrence C.W. Maher V.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4186-4191Crossref PubMed Scopus (162) Google Scholar) and Polη (19Lehman A.R. Kirk-Bell S. Arlett C.F. Paterson M.C. Lohman P.H. de Weerd-Kastelein E.A. Bootsma D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 219-223Crossref PubMed Scopus (523) Google Scholar, 20Maher V.M. Ouellette L.M. Curren R.D. McCormick J.J. Nature. 1976; 261: 593-595Crossref PubMed Scopus (266) Google Scholar, 21McGregor W.G. Wei D. Maher V.M. McCormick J.J. Mol. Cell. Biol. 1999; 19: 147-154Crossref PubMed Scopus (68) Google Scholar, 22Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (384) Google Scholar) are indeed involved in lesion bypass in cells. Lesion bypass can be error-free as a result of insertion of the correct nucleotide opposite the lesion or error-prone as the result of insertion of an incorrect nucleotide opposite the lesion. Both error-free and error-prone nucleotide insertions have been observed with the Y family polymerases depending on the specific lesion and the specific polymerase (reviewed in Refs. 10Wang Z. Mutat. Res. 2001; 486: 59-70Crossref PubMed Scopus (85) Google Scholar, 11Wang Z. Mol. Interv. 2001; 1: 269-281PubMed Google Scholar, 12Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar).Biochemical studies of purified human Polμ have uncovered a unique property that has never been observed with any other polymerases studied so far (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). Human Polμ is highly prone to frameshift DNA synthesis (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). At single-nucleotide repeat sequences, DNA synthesis by human Polμ is mediated mainly by a deletion mechanism because of primer-template realignment before synthesis (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). Furthermore, when the primer 3′ end contains one or a few mismatches, human Polμ can promote primer-template realignment such that the primer 3′ end can find its complementary sequences on the template several nucleotides downstream, achieving microhomology search and microhomology pairing (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). These striking biochemical properties led Zhang et al.(23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar) to propose that Polμ may be involved in nonhomologous end joining (NHEJ) for double-strand DNA repair. The biochemical properties of human Polμ ruled out a significant role for this polymerase in somatic hypermutation during immunoglobulin development.One important cause of DNA double-strand breaks is DNA damage. It is conceivable that some damaged sites may contain clustered lesions or that base damage may be contained near some double-strand DNA breaks. Under those circumstances, Polμ would encounter DNA base damage while performing microhomology search and pairing, as well as DNA synthesis, during NHEJ. Hence, we asked whether Polμ is capable of translesion synthesis. In this report, we demonstrate that human Polμ indeed possesses efficient lesion bypass activities in response to very different types of DNA damage, ranging from simple base modifications and baseless sites to bulky chemical DNA adducts andcis-syn TT dimer of UV radiation. Although in vitro bypass of a template TT dimer is achieved by human Polμ in an error-free manner, bypass of the other tested lesions is mediated by a deletion mechanism that effectively avoids copying the damaged template base through primer realignment. These findings provide new insights into the biochemistry of human Polμ and show that efficient translesion synthesis activity is not strictly confined to Y family polymerases.DISCUSSIONPreviously, we proposed that Polμ may be involved in NHEJ of double-strand DNA breaks through its microhomology searching and pairing activities (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). Most recently, Mahajan et al. (33Mahajan K.N. Nick McElhinny S.A. Mitchell B.S. Ramsden D.A. Mol. Cell. Biol. 2002; 22: 5194-5202Crossref PubMed Scopus (245) Google Scholar) reported that cellular levels of human Polμ protein are increased by ionizing radiation, and that Polμ is associated with the NHEJ proteins Ku and XRCC4-ligase IV, further supporting a role of this polymerase in NHEJ. In this study, we found that human Polμ possesses DNA lesion bypass activities in response to various types of DNA damage. Thus, efficient translesion synthesis activity is not strictly limited to the Y family of DNA polymerases. Under similar experimental conditions, we did not detect any lesion bypass activities of purified human Polβ (Fig. 7), except for 8-oxoguanine (34Zhang Y. Yuan F., Wu, X. Taylor J.-S. Wang Z. Nucleic Acids Res. 2001; 29: 928-935Crossref PubMed Google Scholar), which is a miscoding rather than a strong blocking lesion (35Shibutani S. Takeshita M. Grollman A.P. Nature. 1991; 349: 431-434Crossref PubMed Scopus (2023) Google Scholar). Because Polμ and Polβ share sequence homologies and they both belong to the X family of DNA polymerases (1Dominguez O. Ruiz J.F. Lain de Lera T. Garcia-Diaz M. Gonzalez M.A. Kirchhoff T. Martinez A.C. Bernad A. Blanco L. EMBO J. 2000; 19: 1731-1742Crossref PubMed Google Scholar, 2Aoufouchi S. Flatter E. Dahan A. Faili A. Bertocci B. Storck S. Delbos F. Cocea L. Gupta N. Weill J.C. Reynaud C.A. Nucleic Acids Res. 2000; 28: 3684-3693Crossref PubMed Google Scholar), the lesion bypass activity of human Polμ appears to be unique among X family members.In response to the template AP site 8-oxoguanine, 1,N 6-ethenoadenine, AAF-adducted guanine, and (+)- and (−)-trans-anti-benzo[a]pyrene-N 2-dG, human Polμ bypasses the lesion predominantly by a deletion mechanism. The specificity of nucleotide incorporation during translesion synthesis indicates that deletion is a result of primer realignment. Because these DNA lesions differ dramatically in structure, we propose that bypass of these lesions by human Polμ may be achieved by looping out the template lesion, thus avoiding a direct copying of the damaged template base. The exact deletion size appears to depend on the sequence context of the lesion. For example, if the primer 3′ end can pair with a template base 5′ to the lesion, such realignment would be preferred by human Polμ. Thus, when human Polμ encounters a lesion, if the coding capacity of the modified base is lost or significantly altered, Polμ simply realigns the primer-template strands to continue DNA synthesis by skipping the lesion. Most recently, it was reported that human cell extracts supplemented with purified human Polμ are able to extend a primer from opposite an AAF-adducted guanine by adding a ladder of as many as 15 guanines in an apparently nontemplated reaction (36Duvauchelle J.-B. Blance L. Fuchs R.P.P. Cordonnier A.M. Nucleic Acids Res. 2002; 30: 2061-2067Crossref PubMed Google Scholar). This activity is different from the lesion bypass activity of human Polμ reported here, and its functional significance remains unknown.On the basis of mutation spectra of several DNA lesions, base substitutions rather than deletions appear to be the major mutational events in mammalian cells (37Moriya M. Spiegel S. Fernandes A. Amin S. Liu T. Geacintov N. Grollman A.P. Biochemistry. 1996; 35: 16646-16651Crossref PubMed Scopus (122) Google Scholar, 38Gentil A. Cabral-Neto J.B. Mariage-Samson R. Margot A. Imbach J.L. Rayner B. Sarasin A. J. Mol. Biol. 1992; 227: 981-984Crossref PubMed Scopus (93) Google Scholar, 39Shibutani S. Suzuki N. Grollman A.P. Biochemistry. 1998; 37: 12034-12041Crossref PubMed Scopus (61) Google Scholar, 40Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). Therefore, the prevailing deletion mechanism of lesion bypass by human Polμ suggests that this polymerase is unlikely to play a major role in translesion synthesis during replication in normal cells and under normal growth conditions. It is now clear that single nucleotide repeats, mismatched primer 3′ ends, and many DNA lesions greatly promote the primer-template realignment by human Polμ. These biochemical properties support the role of Polμ in NHEJ. Furthermore, the lesion bypass activities of Polμ would make it possible for this polymerase to perform microhomology search, microhomology pairing, and DNA synthesis during NHEJ even in the presence of base lesions. After NHEJ is complete, the base lesion can then be removed by an excision repair mechanism.Remarkably, the effective bypass of a cis-syn TT dimer by human Polμ is error-free. Because no template TT sequence is present anywhere 5′ to the lesion or near the lesion on the 3′ side (Fig. 6), AA insertion during the bypass must result from the direct copying of the TT dimer by human Polμ. A template TT (6-4) photoproduct, however, completely blocks human Polμ. It is possible that because of covalent linkage between the two thymine bases, the TT dimer and the TT (6-4) photoproduct may not be flexible enough to allow loop-out by human Polμ. Unlike the TT dimer, the TT (6-4) photoproduct may be too distorting to DNA structure to allow Polμ nucleotide insertion opposite the lesion. The only other eukaryotic DNA polymerase known to perform error-free bypass of a TT dimer is Polη (22Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (384) Google Scholar, 41Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (692) Google Scholar). Human xeroderma pigmentosum variant cells that lack Polη activity are sensitive to and hypermutable by UV radiation (19–21,42); thus they establish an important in vivo role for this polymerase in error-free bypass of UV lesions. However, it is not known how Polη would respond to other cyclobutane pyrimidine dimers such as the C-containing dimers. Therefore, it is unknown to what extent loss of the TT dimer bypass by Polη contributes to UV-induced sensitivity and mutagenesis in xeroderma pigmentosum variant cells. With this uncertainty, we are unable to assess at the present time the in vivo importance of the error-free TT dimer bypass by Polμ. Nevertheless, our results raised the possibility that Polμ may participate in the error-free bypass of TT dimers in cells, especially when the Polη function is compromised, as in the case of the xeroderma pigmentosum variant cells. DNA polymerase μ (Polμ) 1The abbreviations used are: Pol, DNA polymerase; BPDE, benzo[a]pyrene-trans-7,8-dihydrodiol-9, 10-epoxide; AAF, N-2-acetylaminofluorene; AP, apurinic/apyrimidinic; NHEJ, nonhomologous end joining 1The abbreviations used are: Pol, DNA polymerase; BPDE, benzo[a]pyrene-trans-7,8-dihydrodiol-9, 10-epoxide; AAF, N-2-acetylaminofluorene; AP, apurinic/apyrimidinic; NHEJ, nonhomologous end joiningis a newly discovered member of the X family polymerases (1Dominguez O. Ruiz J.F. Lain de Lera T. Garcia-Diaz M. Gonzalez M.A. Kirchhoff T. Martinez A.C. Bernad A. Blanco L. EMBO J. 2000; 19: 1731-1742Crossref PubMed Google Scholar, 2Aoufouchi S. Flatter E. Dahan A. Faili A. Bertocci B. Storck S. Delbos F. Cocea L. Gupta N. Weill J.C. Reynaud C.A. Nucleic Acids Res. 2000; 28: 3684-3693Crossref PubMed Google Scholar). Additional members in this family include Polβ, Polλ, and terminal deoxynucleotidyltransferase (1Dominguez O. Ruiz J.F. Lain de Lera T. Garcia-Diaz M. Gonzalez M.A. Kirchhoff T. Martinez A.C. Bernad A. Blanco L. EMBO J. 2000; 19: 1731-1742Crossref PubMed Google Scholar, 2Aoufouchi S. Flatter E. Dahan A. Faili A. Bertocci B. Storck S. Delbos F. Cocea L. Gupta N. Weill J.C. Reynaud C.A. Nucleic Acids Res. 2000; 28: 3684-3693Crossref PubMed Google Scholar, 3Garcia-Diaz M. Dominguez O. Lopez-Fernandez L.A. de Lera L.T. Saniger M.L. Ruiz J.F. Parraga M. Garcia-Ortiz M.J. Kirchhoff T. del Mazo J. Bernad A. Blanco L. J. Mol. Biol. 2000; 301: 851-867Crossref PubMed Scopus (246) Google Scholar). During base excision repair in higher eukaryotes, Polβ is a major repair synthesis polymerase (4Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 5Wood R.D. Shivji M.K. Carcinogenesis. 1997; 18: 605-610Crossref PubMed Scopus (144) Google Scholar, 6Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (264) Google Scholar). Terminal deoxynucleotidyltransferase catalyzes nucleotide additions to DNA in a template-independent manner (7Chang L.M. Bollum F.J. CRC Crit. Rev. Biochem. 1986; 21: 27-52Crossref PubMed Scopus (60) Google Scholar, 8Bentolila L.A. Fanton d'Andon M. Nguyen Q.T. Martinez O. Rougeon F. Doyen N. EMBO J. 1995; 14: 4221-4229Crossref PubMed Scopus (44) Google Scholar). This enzyme functions during V(D)J recombination of the immunoglobulin genes and T-cell receptor genes and is restricted to lymphoid tissues (7Chang L.M. Bollum F.J. CRC Crit. Rev. Biochem. 1986; 21: 27-52Crossref PubMed Scopus (60) Google Scholar, 8Bentolila L.A. Fanton d'Andon M. Nguyen Q.T. Martinez O. Rougeon F. Doyen N. EMBO J. 1995; 14: 4221-4229Crossref PubMed Scopus (44) Google Scholar, 9Weaver D.T. Adv. Immunol. 1995; 58: 29-85Crossref PubMed Scopus (59) Google Scholar). Cellular functions of Polλ and Polμ have not been clearly defined. Although the biochemical activities of the X family DNA polymerases appear to be quite diverse, all of the Y family DNA polymerases share a common biochemical activity: synthesis opposite DNA lesions (reviewed in Refs. 10Wang Z. Mutat. Res. 2001; 486: 59-70Crossref PubMed Scopus (85) Google Scholar, 11Wang Z. Mol. Interv. 2001; 1: 269-281PubMed Google Scholar, 12Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13Friedberg E.C. Fischhaber P.L. Kisker C. Cell. 2001; 107: 9-12Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In eukaryotes, the Y family consists of REV1 and DNA polymerases η, ι, and κ (14Ohmori H. Friedberg E.C. Fuchs R.P.P. Goodman M.F. Hanaoka F. Hinkle D. Kunkel T.A. Lawrence C.W. Livneh Z. Nohmi T. Prakash L. Prakash S. Todo T. Walker G.C. Wang Z. Woodgate R. Mol. Cell. 2001; 8: 7-8Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar). Thus, it is generally believed that a major function of the Y family DNA polymerases is to copy damaged sites of DNA during replication, a cellular process referred to as lesion bypass or translesion synthesis. Genetic studies indicate that REV1 (15Larimer F.W. Perry J.R. Hardigree A.A. J. Bacteriol. 1989; 171: 230-237Crossref PubMed Google Scholar, 16Rajpal D.K., Wu, X. Wang Z. Mutat. Res. 2000; 461: 133-143Crossref PubMed Scopus (37) Google Scholar, 17Nelson J.R. Gibbs P.E. Nowicka A.M. Hinkle D.C. Lawrence C.W. Mol. Microbiol. 2000; 37: 549-554Crossref PubMed Scopus (183) Google Scholar, 18Gibbs P.E. Wang X.D., Li, Z. McManus T.P. McGregor W.G. Lawrence C.W. Maher V.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4186-4191Crossref PubMed Scopus (162) Google Scholar) and Polη (19Lehman A.R. Kirk-Bell S. Arlett C.F. Paterson M.C. Lohman P.H. de Weerd-Kastelein E.A. Bootsma D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 219-223Crossref PubMed Scopus (523) Google Scholar, 20Maher V.M. Ouellette L.M. Curren R.D. McCormick J.J. Nature. 1976; 261: 593-595Crossref PubMed Scopus (266) Google Scholar, 21McGregor W.G. Wei D. Maher V.M. McCormick J.J. Mol. Cell. Biol. 1999; 19: 147-154Crossref PubMed Scopus (68) Google Scholar, 22Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (384) Google Scholar) are indeed involved in lesion bypass in cells. Lesion bypass can be error-free as a result of insertion of the correct nucleotide opposite the lesion or error-prone as the result of insertion of an incorrect nucleotide opposite the lesion. Both error-free and error-prone nucleotide insertions have been observed with the Y family polymerases depending on the specific lesion and the specific polymerase (reviewed in Refs. 10Wang Z. Mutat. Res. 2001; 486: 59-70Crossref PubMed Scopus (85) Google Scholar, 11Wang Z. Mol. Interv. 2001; 1: 269-281PubMed Google Scholar, 12Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Biochemical studies of purified human Polμ have uncovered a unique property that has never been observed with any other polymerases studied so far (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). Human Polμ is highly prone to frameshift DNA synthesis (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). At single-nucleotide repeat sequences, DNA synthesis by human Polμ is mediated mainly by a deletion mechanism because of primer-template realignment before synthesis (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). Furthermore, when the primer 3′ end contains one or a few mismatches, human Polμ can promote primer-template realignment such that the primer 3′ end can find its complementary sequences on the template several nucleotides downstream, achieving microhomology search and microhomology pairing (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). These striking biochemical properties led Zhang et al.(23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar) to propose that Polμ may be involved in nonhomologous end joining (NHEJ) for double-strand DNA repair. The biochemical properties of human Polμ ruled out a significant role for this polymerase in somatic hypermutation during immunoglobulin development. One important cause of DNA double-strand breaks is DNA damage. It is conceivable that some damaged sites may contain clustered lesions or that base damage may be contained near some double-strand DNA breaks. Under those circumstances, Polμ would encounter DNA base damage while performing microhomology search and pairing, as well as DNA synthesis, during NHEJ. Hence, we asked whether Polμ is capable of translesion synthesis. In this report, we demonstrate that human Polμ indeed possesses efficient lesion bypass activities in response to very different types of DNA damage, ranging from simple base modifications and baseless sites to bulky chemical DNA adducts andcis-syn TT dimer of UV radiation. Although in vitro bypass of a template TT dimer is achieved by human Polμ in an error-free manner, bypass of the other tested lesions is mediated by a deletion mechanism that effectively avoids copying the damaged template base through primer realignment. These findings provide new insights into the biochemistry of human Polμ and show that efficient translesion synthesis activity is not strictly confined to Y family polymerases. DISCUSSIONPreviously, we proposed that Polμ may be involved in NHEJ of double-strand DNA breaks through its microhomology searching and pairing activities (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). Most recently, Mahajan et al. (33Mahajan K.N. Nick McElhinny S.A. Mitchell B.S. Ramsden D.A. Mol. Cell. Biol. 2002; 22: 5194-5202Crossref PubMed Scopus (245) Google Scholar) reported that cellular levels of human Polμ protein are increased by ionizing radiation, and that Polμ is associated with the NHEJ proteins Ku and XRCC4-ligase IV, further supporting a role of this polymerase in NHEJ. In this study, we found that human Polμ possesses DNA lesion bypass activities in response to various types of DNA damage. Thus, efficient translesion synthesis activity is not strictly limited to the Y family of DNA polymerases. Under similar experimental conditions, we did not detect any lesion bypass activities of purified human Polβ (Fig. 7), except for 8-oxoguanine (34Zhang Y. Yuan F., Wu, X. Taylor J.-S. Wang Z. Nucleic Acids Res. 2001; 29: 928-935Crossref PubMed Google Scholar), which is a miscoding rather than a strong blocking lesion (35Shibutani S. Takeshita M. Grollman A.P. Nature. 1991; 349: 431-434Crossref PubMed Scopus (2023) Google Scholar). Because Polμ and Polβ share sequence homologies and they both belong to the X family of DNA polymerases (1Dominguez O. Ruiz J.F. Lain de Lera T. Garcia-Diaz M. Gonzalez M.A. Kirchhoff T. Martinez A.C. Bernad A. Blanco L. EMBO J. 2000; 19: 1731-1742Crossref PubMed Google Scholar, 2Aoufouchi S. Flatter E. Dahan A. Faili A. Bertocci B. Storck S. Delbos F. Cocea L. Gupta N. Weill J.C. Reynaud C.A. Nucleic Acids Res. 2000; 28: 3684-3693Crossref PubMed Google Scholar), the lesion bypass activity of human Polμ appears to be unique among X family members.In response to the template AP site 8-oxoguanine, 1,N 6-ethenoadenine, AAF-adducted guanine, and (+)- and (−)-trans-anti-benzo[a]pyrene-N 2-dG, human Polμ bypasses the lesion predominantly by a deletion mechanism. The specificity of nucleotide incorporation during translesion synthesis indicates that deletion is a result of primer realignment. Because these DNA lesions differ dramatically in structure, we propose that bypass of these lesions by human Polμ may be achieved by looping out the template lesion, thus avoiding a direct copying of the damaged template base. The exact deletion size appears to depend on the sequence context of the lesion. For example, if the primer 3′ end can pair with a template base 5′ to the lesion, such realignment would be preferred by human Polμ. Thus, when human Polμ encounters a lesion, if the coding capacity of the modified base is lost or significantly altered, Polμ simply realigns the primer-template strands to continue DNA synthesis by skipping the lesion. Most recently, it was reported that human cell extracts supplemented with purified human Polμ are able to extend a primer from opposite an AAF-adducted guanine by adding a ladder of as many as 15 guanines in an apparently nontemplated reaction (36Duvauchelle J.-B. Blance L. Fuchs R.P.P. Cordonnier A.M. Nucleic Acids Res. 2002; 30: 2061-2067Crossref PubMed Google Scholar). This activity is different from the lesion bypass activity of human Polμ reported here, and its functional significance remains unknown.On the basis of mutation spectra of several DNA lesions, base substitutions rather than deletions appear to be the major mutational events in mammalian cells (37Moriya M. Spiegel S. Fernandes A. Amin S. Liu T. Geacintov N. Grollman A.P. Biochemistry. 1996; 35: 16646-16651Crossref PubMed Scopus (122) Google Scholar, 38Gentil A. Cabral-Neto J.B. Mariage-Samson R. Margot A. Imbach J.L. Rayner B. Sarasin A. J. Mol. Biol. 1992; 227: 981-984Crossref PubMed Scopus (93) Google Scholar, 39Shibutani S. Suzuki N. Grollman A.P. Biochemistry. 1998; 37: 12034-12041Crossref PubMed Scopus (61) Google Scholar, 40Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). Therefore, the prevailing deletion mechanism of lesion bypass by human Polμ suggests that this polymerase is unlikely to play a major role in translesion synthesis during replication in normal cells and under normal growth conditions. It is now clear that single nucleotide repeats, mismatched primer 3′ ends, and many DNA lesions greatly promote the primer-template realignment by human Polμ. These biochemical properties support the role of Polμ in NHEJ. Furthermore, the lesion bypass activities of Polμ would make it possible for this polymerase to perform microhomology search, microhomology pairing, and DNA synthesis during NHEJ even in the presence of base lesions. After NHEJ is complete, the base lesion can then be removed by an excision repair mechanism.Remarkably, the effective bypass of a cis-syn TT dimer by human Polμ is error-free. Because no template TT sequence is present anywhere 5′ to the lesion or near the lesion on the 3′ side (Fig. 6), AA insertion during the bypass must result from the direct copying of the TT dimer by human Polμ. A template TT (6-4) photoproduct, however, completely blocks human Polμ. It is possible that because of covalent linkage between the two thymine bases, the TT dimer and the TT (6-4) photoproduct may not be flexible enough to allow loop-out by human Polμ. Unlike the TT dimer, the TT (6-4) photoproduct may be too distorting to DNA structure to allow Polμ nucleotide insertion opposite the lesion. The only other eukaryotic DNA polymerase known to perform error-free bypass of a TT dimer is Polη (22Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (384) Google Scholar, 41Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (692) Google Scholar). Human xeroderma pigmentosum variant cells that lack Polη activity are sensitive to and hypermutable by UV radiation (19–21,42); thus they establish an important in vivo role for this polymerase in error-free bypass of UV lesions. However, it is not known how Polη would respond to other cyclobutane pyrimidine dimers such as the C-containing dimers. Therefore, it is unknown to what extent loss of the TT dimer bypass by Polη contributes to UV-induced sensitivity and mutagenesis in xeroderma pigmentosum variant cells. With this uncertainty, we are unable to assess at the present time the in vivo importance of the error-free TT dimer bypass by Polμ. Nevertheless, our results raised the possibility that Polμ may participate in the error-free bypass of TT dimers in cells, especially when the Polη function is compromised, as in the case of the xeroderma pigmentosum variant cells. Previously, we proposed that Polμ may be involved in NHEJ of double-strand DNA breaks through its microhomology searching and pairing activities (23Zhang Y., Wu, X. Yuan F. Xie Z. Wang Z. Mol. Cell. Biol. 2001; 21: 7995-8006Crossref PubMed Scopus (80) Google Scholar). Most recently, Mahajan et al. (33Mahajan K.N. Nick McElhinny S.A. Mitchell B.S. Ramsden D.A. Mol. Cell. Biol. 2002; 22: 5194-5202Crossref PubMed Scopus (245) Google Scholar) reported that cellular levels of human Polμ protein are increased by ionizing radiation, and that Polμ is associated with the NHEJ proteins Ku and XRCC4-ligase IV, further supporting a role of this polymerase in NHEJ. In this study, we found that human Polμ possesses DNA lesion bypass activities in response to various types of DNA damage. Thus, efficient translesion synthesis activity is not strictly limited to the Y family of DNA polymerases. Under similar experimental conditions, we did not detect any lesion bypass activities of purified human Polβ (Fig. 7), except for 8-oxoguanine (34Zhang Y. Yuan F., Wu, X. Taylor J.-S. Wang Z. Nucleic Acids Res. 2001; 29: 928-935Crossref PubMed Google Scholar), which is a miscoding rather than a strong blocking lesion (35Shibutani S. Takeshita M. Grollman A.P. Nature. 1991; 349: 431-434Crossref PubMed Scopus (2023) Google Scholar). Because Polμ and Polβ share sequence homologies and they both belong to the X family of DNA polymerases (1Dominguez O. Ruiz J.F. Lain de Lera T. Garcia-Diaz M. Gonzalez M.A. Kirchhoff T. Martinez A.C. Bernad A. Blanco L. EMBO J. 2000; 19: 1731-1742Crossref PubMed Google Scholar, 2Aoufouchi S. Flatter E. Dahan A. Faili A. Bertocci B. Storck S. Delbos F. Cocea L. Gupta N. Weill J.C. Reynaud C.A. Nucleic Acids Res. 2000; 28: 3684-3693Crossref PubMed Google Scholar), the lesion bypass activity of human Polμ appears to be unique among X family members. In response to the template AP site 8-oxoguanine, 1,N 6-ethenoadenine, AAF-adducted guanine, and (+)- and (−)-trans-anti-benzo[a]pyrene-N 2-dG, human Polμ bypasses the lesion predominantly by a deletion mechanism. The specificity of nucleotide incorporation during translesion synthesis indicates that deletion is a result of primer realignment. Because these DNA lesions differ dramatically in structure, we propose that bypass of these lesions by human Polμ may be achieved by looping out the template lesion, thus avoiding a direct copying of the damaged template base. The exact deletion size appears to depend on the sequence context of the lesion. For example, if the primer 3′ end can pair with a template base 5′ to the lesion, such realignment would be preferred by human Polμ. Thus, when human Polμ encounters a lesion, if the coding capacity of the modified base is lost or significantly altered, Polμ simply realigns the primer-template strands to continue DNA synthesis by skipping the lesion. Most recently, it was reported that human cell extracts supplemented with purified human Polμ are able to extend a primer from opposite an AAF-adducted guanine by adding a ladder of as many as 15 guanines in an apparently nontemplated reaction (36Duvauchelle J.-B. Blance L. Fuchs R.P.P. Cordonnier A.M. Nucleic Acids Res. 2002; 30: 2061-2067Crossref PubMed Google Scholar). This activity is different from the lesion bypass activity of human Polμ reported here, and its functional significance remains unknown. On the basis of mutation spectra of several DNA lesions, base substitutions rather than deletions appear to be the major mutational events in mammalian cells (37Moriya M. Spiegel S. Fernandes A. Amin S. Liu T. Geacintov N. Grollman A.P. Biochemistry. 1996; 35: 16646-16651Crossref PubMed Scopus (122) Google Scholar, 38Gentil A. Cabral-Neto J.B. Mariage-Samson R. Margot A. Imbach J.L. Rayner B. Sarasin A. J. Mol. Biol. 1992; 227: 981-984Crossref PubMed Scopus (93) Google Scholar, 39Shibutani S. Suzuki N. Grollman A.P. Biochemistry. 1998; 37: 12034-12041Crossref PubMed Scopus (61) Google Scholar, 40Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). Therefore, the prevailing deletion mechanism of lesion bypass by human Polμ suggests that this polymerase is unlikely to play a major role in translesion synthesis during replication in normal cells and under normal growth conditions. It is now clear that single nucleotide repeats, mismatched primer 3′ ends, and many DNA lesions greatly promote the primer-template realignment by human Polμ. These biochemical properties support the role of Polμ in NHEJ. Furthermore, the lesion bypass activities of Polμ would make it possible for this polymerase to perform microhomology search, microhomology pairing, and DNA synthesis during NHEJ even in the presence of base lesions. After NHEJ is complete, the base lesion can then be removed by an excision repair mechanism. Remarkably, the effective bypass of a cis-syn TT dimer by human Polμ is error-free. Because no template TT sequence is present anywhere 5′ to the lesion or near the lesion on the 3′ side (Fig. 6), AA insertion during the bypass must result from the direct copying of the TT dimer by human Polμ. A template TT (6-4) photoproduct, however, completely blocks human Polμ. It is possible that because of covalent linkage between the two thymine bases, the TT dimer and the TT (6-4) photoproduct may not be flexible enough to allow loop-out by human Polμ. Unlike the TT dimer, the TT (6-4) photoproduct may be too distorting to DNA structure to allow Polμ nucleotide insertion opposite the lesion. The only other eukaryotic DNA polymerase known to perform error-free bypass of a TT dimer is Polη (22Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (384) Google Scholar, 41Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (692) Google Scholar). Human xeroderma pigmentosum variant cells that lack Polη activity are sensitive to and hypermutable by UV radiation (19–21,42); thus they establish an important in vivo role for this polymerase in error-free bypass of UV lesions. However, it is not known how Polη would respond to other cyclobutane pyrimidine dimers such as the C-containing dimers. Therefore, it is unknown to what extent loss of the TT dimer bypass by Polη contributes to UV-induced sensitivity and mutagenesis in xeroderma pigmentosum variant cells. With this uncertainty, we are unable to assess at the present time the in vivo importance of the error-free TT dimer bypass by Polμ. Nevertheless, our results raised the possibility that Polμ may participate in the error-free bypass of TT dimers in cells, especially when the Polη function is compromised, as in the case of the xeroderma pigmentosum variant cells.
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