DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.
References
[1]
Lange SS, Takata K, Wood RD. DNA polymerases and cancer. Nat Rev Cancer. 2011;11(2):96–110. doi: 10.1038/nrc2998 pmid:21258395; PubMed Central PMCID: PMC3739438.
[2]
Shen X, Jun S, O'Neal LE, Sonoda E, Bemark M, Sale JE, et al. REV3 and REV1 play major roles in recombination-independent repair of DNA interstrand cross-links mediated by monoubiquitinated proliferating cell nuclear antigen (PCNA). J Biol Chem. 2006;281(20):13869–72. pmid:16571727. doi: 10.1074/jbc.c600071200
[3]
Schenten D, Kracker S, Esposito G, Franco S, Klein U, Murphy M, et al. Polζ ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability. J Exp Med. 2009;206(2):477–90. Epub 2009/02/11. doi: 10.1084/jem.20080669 pmid:19204108.
[4]
Hashimoto K, Cho Y, Yang IY, Akagi J, Ohashi E, Tateishi S, et al. The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site. J Biol Chem. 2012;287(12):9613–22. doi: 10.1074/jbc.M111.331728 pmid:22303021; PubMed Central PMCID: PMC3308759.
[5]
Shachar S, Ziv O, Avkin S, Adar S, Wittschieben J, Reissner T, et al. Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals. EMBO J. 2009;28:383–93. Epub 2009/01/21. doi: 10.1038/emboj.2008.281 pmid:19153606.
[6]
Gan GN, Wittschieben JP, Wittschieben B？, Wood RD. DNA polymerase ζ (polζ) in higher eukaryotes. Cell Res. 2008;18(1):174–83. pmid:18157155. doi: 10.1038/cr.2007.117
[7]
Lange SS, Bedford E, Reh S, Wittschieben JP, Carbajal S, Kusewitt DF, et al. Dual role for mammalian DNA polymerase ζ in maintaining genome stability and proliferative responses. Proc Natl Acad Sci USA. 2013;110(8):E687–E96. doi: 10.1073/pnas.1217425110 pmid:23386725; PubMed Central PMCID: PMC3581960.
[8]
Lange SS, Wittschieben JP, Wood RD. DNA polymerase ζ is required for proliferation of normal mammalian cells. Nucleic Acids Res. 2012;40(10):4473–82. Epub 2012/02/10. doi: 10.1093/nar/gks054 pmid:22319213; PubMed Central PMCID: PMC3378892.
[9]
Wittschieben JP, Gollin SM, Reshmi SC, Wood RD. Loss of DNA polymerase ζ causes chromosomal instability in mammalian cells. Cancer Res. 2006;66(1):134–42. pmid:16397225. doi: 10.1158/0008-5472.can-05-2982
[10]
Zander L, Bemark M. Immortalized mouse cell lines that lack a functional Rev3 gene are hypersensitive to UV irradiation and cisplatin treatment. DNA Repair (Amst). 2004;3(7):743–52. pmid:15177183. doi: 10.1016/j.dnarep.2004.03.031
[11]
Goff JP, Shields DS, Seki M, Choi S, Epperly MW, Dixon T, et al. Lack of DNA polymerase θ (POLQ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation. Radiat Res. 2009;172(2):165–74. Epub 2009/07/28. doi: 10.1667/RR1598.1 pmid:19630521; PubMed Central PMCID: PMC2742993.
[12]
Xu X, Xie K, Zhang XQ, Pridgen EM, Park GY, Cui DS, et al. Enhancing tumor cell response to chemotherapy through nanoparticle-mediated codelivery of siRNA and cisplatin prodrug. Proc Natl Acad Sci USA. 2013;110(46):18638–43. doi: 10.1073/pnas.1303958110 pmid:24167294; PubMed Central PMCID: PMC3832000.
[13]
Kikuchi S, Hara K, Shimizu T, Sato M, Hashimoto H. Structural basis of recruitment of DNA polymerase ζ by interaction between REV1 and REV7 proteins. J Biol Chem. 2012;287(40):33847–52. doi: 10.1074/jbc.M112.396838 pmid:22859296; PubMed Central PMCID: PMC3460479.
[14]
Hanafusa T, Habu T, Tomida J, Ohashi E, Murakumo Y, Ohmori H. Overlapping in short motif sequences for binding to human REV7 and MAD2 proteins. Genes Cells. 2010. doi: 10.1111/j.1365-2443.2009.01380.x pmid:20088965.
[15]
Wojtaszek J, Liu J, D'Souza S, Wang S, Xue Y, Walker GC, et al. Multifaceted recognition of vertebrate Rev1 by translesion polymerases ζ and κ. J Biol Chem. 2012;287(31):26400–8. Epub 2012/06/16. doi: 10.1074/jbc.M112.380998 pmid:22700975; PubMed Central PMCID: PMC3406723.
[16]
Guo C, Fischhaber PL, Luk-Paszyc MJ, Masuda Y, Zhou J, Kamiya K, et al. Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis. EMBO J. 2003;22(24):6621–30. pmid:14657033. doi: 10.1093/emboj/cdg626
[17]
Wojtaszek J, Lee CJ, D'Souza S, Minesinger B, Kim H, D'Andrea AD, et al. Structural basis of Rev1-mediated assembly of a quaternary vertebrate translesion polymerase complex consisting of Rev1, heterodimeric polymerase (Pol) ζ, and Pol κ. J Biol Chem. 2012;287(40):33836–46. Epub 2012/08/04. doi: 10.1074/jbc.M112.394841 pmid:22859295; PubMed Central PMCID: PMC3460478.
[18]
Pozhidaeva A, Pustovalova Y, D'Souza S, Bezsonova I, Walker GC, Korzhnev DM. NMR structure and dynamics of the C-terminal domain from human Rev1 and its complex with Rev1 interacting region of DNA polymerase η. Biochemistry. 2012;51(27):5506–20. doi: 10.1021/bi300566z pmid:22691049; PubMed Central PMCID: PMC3732116.
[19]
Tomida J, Takata K, Lange SS, Schibler AC, Yousefzadeh MJ, Bhetawal S, et al. REV7 is essential for DNA damage tolerance via two REV3L binding sites in mammalian DNA polymerase ζ. Nucleic Acids Res. 2015;43(2):1000–11. doi: 10.1093/nar/gku1385 pmid:25567983; PubMed Central PMCID: PMC4333420.
[20]
Watanabe N, Mii S, Asai N, Asai M, Niimi K, Ushida K, et al. The Rev7 Subunit of DNA Polymerase ζ Is Essential for Primordial Germ Cell Maintenance in the Mouse. The Journal of biological chemistry. 2013. Epub 2013/03/07. doi: 10.1074/jbc.M112.421966 pmid:23463509.
[21]
Pirouz M, Pilarski S, Kessel M. A critical function of Mad2l2 in primordial germ cell development of mice. PLoS Genet. 2013;9(8):e1003712. doi: 10.1371/journal.pgen.1003712 pmid:24009519; PubMed Central PMCID: PMC3757036.
[22]
Gibbs PE, McGregor WG, Maher VM, Nisson P, Lawrence CW. A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ. Proc Natl Acad Sci USA. 1998;95(12):6876–80. pmid:9618506 doi: 10.1073/pnas.95.12.6876
[23]
Netz DJ, Stith CM, Stumpfig M, Kopf G, Vogel D, Genau HM, et al. Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes. Nat Chem Biol. 2012;8(1):125–32. Epub 2011/11/29. doi: 10.1038/nchembio.721 pmid:22119860; PubMed Central PMCID: PMC3241888.
[24]
Lee YS, Gregory MT, Yang W. Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass. Proc Natl Acad Sci U S A. 2014;111(8):2954–9. doi: 10.1073/pnas.1324001111 pmid:24449906; PubMed Central PMCID: PMC3939873.
[25]
Johnson RE, Prakash L, Prakash S. Pol31 and Pol32 subunits of yeast DNA polymerase δ are also essential subunits of DNA polymerase ζ. Proc Natl Acad Sci USA. 2012. Epub 2012/06/20. doi: 10.1073/pnas.1206052109 pmid:22711820.
[26]
Baranovskiy AG, Lada AG, Siebler H, Zhang Y, Pavlov YI, Tahirov TH. DNA polymerases δ and ζ switching by sharing the accessory subunits of DNA polymerase δ. J Biol Chem. 2012;287(21):17281–7. Epub 2012/04/03. doi: 10.1074/jbc.M112.351122 pmid:22465957.
[27]
Hirota K, Sonoda E, Kawamoto T, Motegi A, Masutani C, Hanaoka F, et al. Simultaneous Disruption of Two DNA Polymerases, Pol η and Pol ζ, in Avian DT40 Cells Unmasks the Role of Pol η in Cellular Response to Various DNA Lesions. PLoS Genet. 2010;6(10):e1001151. doi: 10.1371/journal.pgen.1001151. pmid:20949111
[28]
Nakatani Y, Ogryzko V. Immunoaffinity purification of mammalian protein complexes. Methods Enzymol. 2003;370:430–44. Epub 2004/01/10. doi: 10.1016/S0076-6879(03)70037-8 pmid:14712665.
[29]
Copeland WC, Wang TS. Mutational analysis of the human DNA polymerase α. The most conserved region in α-like DNA polymerases is involved in metal-specific catalysis. J Biol Chem. 1993;268(15):11028–40. pmid:8496164.
[30]
Polesky AH, Steitz TA, Grindley ND, Joyce CM. Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli. J Biol Chem. 1990;265(24):14579–91. pmid:2201688.
[31]
Gueranger Q, Stary A, Aoufouchi S, Faili A, Sarasin A, Reynaud CA, et al. Role of DNA polymerases η, ι and ζ in UV resistance and UV-induced mutagenesis in a human cell line. DNA Repair (Amst). 2008;7(9):1551–62. Epub 2008/07/01. doi: 10.1016/j.dnarep.2008.05.012 pmid:18586118.
[32]
Schnutgen F, Doerflinger N, Calleja C, Wendling O, Chambon P, Ghyselinck NB. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol. 2003;21(5):562–5. doi: 10.1038/nbt811 pmid:12665802.
[33]
Esposito G, Godindagger I, Klein U, Yaspo ML, Cumano A, Rajewsky K. Disruption of the Rev3l-encoded catalytic subunit of polymerase ζ in mice results in early embryonic lethality. Current biology. 2000;10(19):1221–4. Epub 2000/10/26. pmid:11050393. doi: 10.1016/s0960-9822(00)00726-0
[34]
Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45(9):593–605. Epub 2007/09/18. doi: 10.1002/dvg.20335 pmid:17868096.
[35]
Albertella MR, Green CM, Lehmann AR, O'Connor MJ. A role for polymerase η in the cellular tolerance to cisplatin-induced damage. Cancer Res. 2005;65(21):9799–806. pmid:16267001. doi: 10.1158/0008-5472.can-05-1095
[36]
de Boer J, Donker I, de Wit J, Hoeijmakers JH, Weeda G. Disruption of the mouse xeroderma pigmentosum group D DNA repair/basal transcription gene results in preimplantation lethality. Cancer Res. 1998;58(1):89–94. pmid:9426063.
[37]
Andressoo JO, Mitchell JR, de Wit J, Hoogstraten D, Volker M, Toussaint W, et al. An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria. Cancer Cell. 2006;10(2):121–32. pmid:16904611. doi: 10.1016/j.ccr.2006.05.027
[38]
Winkler GS, Araújo SJ, Fiedler U, Vermeulen W, Coin F, Egly J-M, et al. TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair. J Biol Chem. 2000;275:4258–66. pmid:10660593 doi: 10.1074/jbc.275.6.4258
[39]
Kuper J, Braun C, Elias A, Michels G, Sauer F, Schmitt DR, et al. In TFIIH, XPD Helicase Is Exclusively Devoted to DNA Repair. PLoS Biol. 2014;12(9):e1001954. doi: 10.1371/journal.pbio.1001954 pmid:25268380; PubMed Central PMCID: PMC4182028.
[40]
Ross AL, Simpson LJ, Sale JE. Vertebrate DNA damage tolerance requires the C-terminus but not BRCT or transferase domains of REV1. Nucleic Acids Res. 2005;33(4):1280–9. pmid:15741181. doi: 10.1093/nar/gki279
[41]
Kanemaru Y, Suzuki T, Niimi N, Gruz P, Matsumoto K, Adachi N, et al. Catalytic and non-catalytic roles of DNA polymerase kappa in the protection of human cells against genotoxic stresses. Environ Mol Mutagen. 2015;56(8):650–62. doi: 10.1002/em.21961 pmid:26031400.
[42]
Takezawa J, Aiba N, Kajiwara K, Yamada K. Caffeine abolishes the ultraviolet-induced REV3 translesion replication pathway in mouse cells. Int J Mol Sci. 2011;12(12):8513–29. Epub 2012/01/25. doi: 10.3390/ijms12128513 pmid:22272088; PubMed Central PMCID: PMC3257085.
[43]
Hicks JK, Chute CL, Paulsen MT, Ragland RL, Howlett NG, Gueranger Q, et al. Differential roles for DNA polymerases η, ζ, and REV1 in lesion bypass of intrastrand versus interstrand DNA cross-links. Mol Cell Biol. 2010;30(5):1217–30. Epub 2009/12/24. doi: 10.1128/MCB.00993-09 pmid:20028736; PubMed Central PMCID: PMC2820889.
Sharma S, Shah NA, Joiner AM, Roberts KH, Canman CE. DNA polymerase ζ is a major determinant of resistance to platinum-based chemotherapeutic agents. Mol Pharmacol. 2012;81(6):778–87. doi: 10.1124/mol.111.076828 pmid:22387291; PubMed Central PMCID: PMC3362893.
[46]
Janel-Bintz R, Wagner J, Haracska L, Mah-Becherel MC, Bichara M, Fuchs RP, et al. Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts. PLoS One. 2012;7(4):e36004. doi: 10.1371/journal.pone.0036004 pmid:22558303; PubMed Central PMCID: PMC3338768.
[47]
Faili A, Aoufouchi S, Flatter E, Gueranger Q, Reynaud CA, Weill JC. Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase ι. Nature. 2002;419(6910):944–7. pmid:12410315. doi: 10.1038/nature01117
[48]
Shimizu T, Azuma T, Ishiguro M, Kanjo N, Yamada S, Ohmori H. Normal immunoglobulin gene somatic hypermutation in Pol κ Pol ι double-deficient mice. Immunol Lett. 2005;98(2):259–64. doi: 10.1016/j.imlet.2004.11.022 pmid:15860226.
[49]
Delbos F, Aoufouchi S, Faili A, Weill JC, Reynaud CA. DNA polymerase η is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse. J Exp Med. 2007;204(1):17–23. pmid:17190840. doi: 10.1084/jem.20062131
[50]
Abdulovic AL, Jinks-Robertson S. The in vivo characterization of translesion synthesis across UV-induced lesions in Saccharomyces cerevisiae: insights into Pol ζ- and Pol η-dependent frameshift mutagenesis. Genetics. 2006;172(3):1487–98. doi: 10.1534/genetics.105.052480 pmid:16387871; PubMed Central PMCID: PMC1456278.
[51]
Chatterjee N, Pabla R, Siede W. Role of polymerase η in mitochondrial mutagenesis of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 2013;431(2):270–3. doi: 10.1016/j.bbrc.2012.12.119 pmid:23313845.
[52]
Ohkumo T, Kondo Y, Yokoi M, Tsukamoto T, Yamada A, Sugimoto T, et al. UV-B radiation induces epithelial tumors in mice lacking DNA polymerase η and mesenchymal tumors in mice deficient for DNA polymerase ι. Mol Cell Biol. 2006;26(20):7696–706. doi: 10.1128/MCB.01076-06 pmid:17015482; PubMed Central PMCID: PMC1636855.
[53]
Doles J, Oliver TG, Cameron ER, Hsu G, Jacks T, Walker GC, et al. Suppression of Rev3, the catalytic subunit of Pol ζ, sensitizes drug-resistant lung tumors to chemotherapy. Proc Natl Acad Sci USA. 2010;107(48):20786–91. Epub 2010/11/12. doi: 10.1073/pnas.1011409107 pmid:21068376; PubMed Central PMCID: PMC2996428.
[54]
McDonald JP, Frank EG, Plosky BS, Rogozin IB, Masutani C, Hanaoka F, et al. 129-derived strains of mice are deficient in DNA polymerase ι and have normal immunoglobulin hypermutation. J Exp Med. 2003;198(4):635–43. pmid:12925679. doi: 10.1084/jem.20030767
[55]
Lin W, Wu X, Wang Z. A full-length cDNA of hREV3 is predicted to encode DNA polymerase ζ for damage-induced mutagenesis in humans. Mutat Res. 1999;433(2):89–98. pmid:10102035. doi: 10.1016/s0921-8777(98)00065-2
[56]
Ishiai M, Kitao H, Smogorzewska A, Tomida J, Kinomura A, Uchida E, et al. FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway. Nat Struct Mol Biol. 2008;15(11):1138–46. doi: 10.1038/nsmb.1504 pmid:18931676; PubMed Central PMCID: PMC3293454.
[57]
Takata K, Reh S, Tomida J, Person MD, Wood RD. Human DNA helicase HELQ participates in DNA interstrand crosslink tolerance with ATR and RAD51 paralogs. Nature Communications. 2013;4. doi: 10.1038/ncomms3338 pmid:24005565; PubMed Central PMCID: PMC3778836.
[58]
Saribasak H, Maul RW, Cao Z, Yang WW, Schenten D, Kracker S, et al. DNA polymerase ζ generates tandem mutations in immunoglobulin variable regions. The Journal of experimental medicine. 2012;209(6):1075–81. Epub 2012/05/23. doi: 10.1084/jem.20112234 pmid:22615128; PubMed Central PMCID: PMC3371727.
[59]
Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7(10):R100. doi: 10.1186/gb-2006-7-10-r100 pmid:17076895; PubMed Central PMCID: PMC1794559.
[60]
Wood RD, Mitchell M, Sgouros J, Lindahl T. Human DNA repair genes. Science. 2001;291:1284–9. pmid:11181991 doi: 10.1016/s0002-9394(01)01079-0
[61]
Cantagrel V, Lossi AM, Boulanger S, Depetris D, Mattei MG, Gecz J, et al. Disruption of a new X linked gene highly expressed in brain in a family with two mentally retarded males. J Med Genet. 2004;41(10):736–42. doi: 10.1136/jmg.2004.021626. pmid:15466006
[62]
Singh B, Li X, Owens KM, Vanniarajan A, Liang P, Singh KK. Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome. PLoS One. 2015;10(10):e0140409. doi: 10.1371/journal.pone.0140409 pmid:26462070; PubMed Central PMCID: PMCPMC4604079.
[63]
Emerson JJ, Kaessmann H, Betran E, Long M. Extensive gene traffic on the mammalian X chromosome. Science. 2004;303(5657):537–40. doi: 10.1126/science.1090042 pmid:14739461.
[64]
Chen J, Ma A, Young F, Alt FW. IL-2 receptor alpha chain expression during early B lymphocyte differentiation. Int Immunol. 1994;6(8):1265–8. pmid:7981152. doi: 10.1093/intimm/6.8.1265
