Translesion synthesis polymerases are dispensable for C. elegans reproduction but suppress genome scarring by polymerase theta-mediated end joining

Autoři: Ivo van Bostelen aff001;  Robin van Schendel aff001;  Ron Romeijn aff001;  Marcel Tijsterman aff001
Působiště autorů: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands aff001;  Institute of Biology Leiden, Leiden University, Leiden, the Netherlands aff002
Vyšlo v časopise: Translesion synthesis polymerases are dispensable for C. elegans reproduction but suppress genome scarring by polymerase theta-mediated end joining. PLoS Genet 16(4): e32767. doi:10.1371/journal.pgen.1008759
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008759


Bases within DNA are frequently damaged, producing obstacles to efficient and accurate DNA replication by replicative polymerases. Translesion synthesis (TLS) polymerases, via their ability to catalyze nucleotide additions to growing DNA chains across DNA lesions, promote replication of damaged DNA, thus preventing checkpoint activation, genome instability and cell death. In this study, we used C. elegans to determine the contribution of TLS activity on long-term stability of an animal genome. We monitored and compared the types of mutations that accumulate in REV1, REV3, POLH1 and POLK deficient animals that were grown under unchallenged conditions. We also addressed redundancies in TLS activity by combining all deficiencies. Remarkably, animals that are deficient for all Y-family polymerases as well as animals that have lost all TLS activity are viable and produce progeny, demonstrating that TLS is not essential for animal life. Whole genome sequencing analyses, however, reveal that TLS is needed to prevent genomic scars from accumulating. These scars, which are the product of polymerase theta-mediated end joining (TMEJ), are found overrepresented at guanine bases, consistent with TLS suppressing DNA double-strand breaks (DSBs) from occurring at replication-blocking guanine adducts. We found that in C. elegans, TLS across spontaneous damage is predominantly error free and anti-clastogenic, and thus ensures preservation of genetic information.

Klíčová slova:

Animal genomics – Caenorhabditis elegans – DNA damage – DNA replication – Invertebrate genomics – Mutagenesis – Mutation – Polymerases


1. McCulloch SD, Kunkel TA. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases. Cell research. 2008;18(1):148–61. Epub 2008/01/02. doi: 10.1038/cr.2008.4 18166979; PubMed Central PMCID: PMC3639319.

2. Fishel R. Mismatch repair. The Journal of biological chemistry. 2015;290(44):26395–403. Epub 2015/09/12. doi: 10.1074/jbc.R115.660142 26354434; PubMed Central PMCID: PMC4646297.

3. Barnes DE, Lindahl T. Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annual review of genetics. 2004;38:445–76. Epub 2004/12/01. doi: 10.1146/annurev.genet.38.072902.092448 15568983.

4. Marteijn JA, Lans H, Vermeulen W, Hoeijmakers JH. Understanding nucleotide excision repair and its roles in cancer and ageing. Nature reviews Molecular cell biology. 2014;15(7):465–81. Epub 2014/06/24. doi: 10.1038/nrm3822 24954209.

5. Pilzecker B, Buoninfante OA, Jacobs H. DNA damage tolerance in stem cells, ageing, mutagenesis, disease and cancer therapy. Nucleic acids research. 2019;47(14):7163–81. Epub 2019/06/30. doi: 10.1093/nar/gkz531 31251805; PubMed Central PMCID: PMC6698745.

6. Hirota K, Yoshikiyo K, Guilbaud G, Tsurimoto T, Murai J, Tsuda M, et al. The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ. Nucleic acids research. 2015;43(3):1671–83. Epub 2015/01/30. doi: 10.1093/nar/gkv023 25628356; PubMed Central PMCID: PMC4330384.

7. Jansen JG, Fousteri MI, de Wind N. Send in the clamps: control of DNA translesion synthesis in eukaryotes. Molecular cell. 2007;28(4):522–9. Epub 2007/11/29. doi: 10.1016/j.molcel.2007.11.005 18042449.

8. Prakash S, Johnson RE, Prakash L. Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annual review of biochemistry. 2005;74:317–53. Epub 2005/06/15. doi: 10.1146/annurev.biochem.74.082803.133250 15952890.

9. Sale JE, Lehmann AR, Woodgate R. Y-family DNA polymerases and their role in tolerance of cellular DNA damage. Nature reviews Molecular cell biology. 2012;13(3):141–52. Epub 2012/02/24. doi: 10.1038/nrm3289 22358330; PubMed Central PMCID: PMC3630503.

10. Baranovskiy AG, Lada AG, Siebler HM, Zhang Y, Pavlov YI, Tahirov TH. DNA polymerase δ and ζ switch by sharing accessory subunits of DNA polymerase δ. The Journal of biological chemistry. 2012;287(21):17281–7. Epub 2012/04/03. doi: 10.1074/jbc.M112.351122 22465957; PubMed Central PMCID: PMC3366816.

11. Johnson RE, Prakash L, Prakash S. Pol31 and Pol32 subunits of yeast DNA polymerase δ are also essential subunits of DNA polymerase ζ. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(31):12455–60. Epub 2012/06/20. doi: 10.1073/pnas.1206052109 22711820; PubMed Central PMCID: PMC3411960.

12. Makarova AV, Stodola JL, Burgers PM. A four-subunit DNA polymerase ζ complex containing Pol δ accessory subunits is essential for PCNA-mediated mutagenesis. Nucleic acids research. 2012;40(22):11618–26. Epub 2012/10/16. doi: 10.1093/nar/gks948 23066099; PubMed Central PMCID: PMC3526297.

13. Johnson RE, Washington MT, Haracska L, Prakash S, Prakash L. Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions. Nature. 2000;406(6799):1015–9. Epub 2000/09/13. doi: 10.1038/35023030 10984059.

14. Prakash S, Prakash L. Translesion DNA synthesis in eukaryotes: a one- or two-polymerase affair. Genes & development. 2002;16(15):1872–83. Epub 2002/08/03. doi: 10.1101/gad.1009802 12154119.

15. Gan GN, Wittschieben JP, Wittschieben B, Wood RD. DNA polymerase zeta (pol zeta) in higher eukaryotes. Cell research. 2008;18(1):174–83. Epub 2007/12/25. doi: 10.1038/cr.2007.117 18157155.

16. Roerink SF, Koole W, Stapel LC, Romeijn RJ, Tijsterman M. A broad requirement for TLS polymerases η and κ, and interacting sumoylation and nuclear pore proteins, in lesion bypass during C. elegans embryogenesis. PLoS genetics. 2012;8(6):e1002800. Epub 2012/07/05. doi: 10.1371/journal.pgen.1002800 22761594; PubMed Central PMCID: PMC3386174.

17. Roerink SF, van Schendel R, Tijsterman M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome research. 2014;24(6):954–62. Epub 2014/03/13. doi: 10.1101/gr.170431.113 24614976; PubMed Central PMCID: PMC4032859.

18. van Bostelen I, Tijsterman M. Combined loss of three DNA damage response pathways renders C. elegans intolerant to light. DNA repair. 2017;54:55–62. Epub 2017/05/05. doi: 10.1016/j.dnarep.2017.04.002 28472716.

19. Thompson O, Edgley M, Strasbourger P, Flibotte S, Ewing B, Adair R, et al. The million mutation project: a new approach to genetics in Caenorhabditis elegans. Genome research. 2013;23(10):1749–62. Epub 2013/06/27. doi: 10.1101/gr.157651.113 23800452; PubMed Central PMCID: PMC3787271.

20. 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. The EMBO journal. 2003;22(24):6621–30. Epub 2003/12/06. doi: 10.1093/emboj/cdg626 14657033; PubMed Central PMCID: PMC291821.

21. Lawrence CW. Cellular functions of DNA polymerase zeta and Rev1 protein. Advances in protein chemistry. 2004;69:167–203. Epub 2004/12/14. doi: 10.1016/S0065-3233(04)69006-1 15588843.

22. Pryor JM, Gakhar L, Washington MT. Structure and functional analysis of the BRCT domain of translesion synthesis DNA polymerase Rev1. Biochemistry. 2013;52(1):254–63. Epub 2012/12/18. doi: 10.1021/bi301572z 23240687; PubMed Central PMCID: PMC3580236.

23. Jansen JG, Tsaalbi-Shtylik A, Langerak P, Calléja F, Meijers CM, Jacobs H, et al. The BRCT domain of mammalian Rev1 is involved in regulating DNA translesion synthesis. Nucleic acids research. 2005;33(1):356–65. Epub 2005/01/18. doi: 10.1093/nar/gki189 15653636; PubMed Central PMCID: PMC546167.

24. Larimer FW, Perry JR, Hardigree AA. The REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis. Journal of bacteriology. 1989;171(1):230–7. Epub 1989/01/01. doi: 10.1128/jb.171.1.230-237.1989 2492497; PubMed Central PMCID: PMC209577.

25. Gibbs PE, Wang XD, Li Z, McManus TP, McGregor WG, Lawrence CW, et al. The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(8):4186–91. Epub 2000/04/13. doi: 10.1073/pnas.97.8.4186 10760286; PubMed Central PMCID: PMC18191.

26. Johnson RE, Kondratick CM, Prakash S, Prakash L. hRAD30 mutations in the variant form of xeroderma pigmentosum. Science (New York, NY). 1999;285(5425):263–5. Epub 1999/07/10. doi: 10.1126/science.285.5425.263 10398605.

27. De Stasio E, Lephoto C, Azuma L, Holst C, Stanislaus D, Uttam J. Characterization of revertants of unc-93(e1500) in Caenorhabditis elegans induced by N-ethyl-N-nitrosourea. Genetics. 1997;147(2):597–608. Epub 1997/10/23. 9335597; PubMed Central PMCID: PMC1208182.

28. Greenwald IS, Horvitz HR. unc-93(e1500): A behavioral mutant of Caenorhabditis elegans that defines a gene with a wild-type null phenotype. Genetics. 1980;96(1):147–64. Epub 1980/09/01. 6894129; PubMed Central PMCID: PMC1214286.

29. Kruisselbrink E, Guryev V, Brouwer K, Pontier DB, Cuppen E, Tijsterman M. Mutagenic capacity of endogenous G4 DNA underlies genome instability in FANCJ-defective C. elegans. Current biology: CB. 2008;18(12):900–5. Epub 2008/06/10. doi: 10.1016/j.cub.2008.05.013 18538569.

30. Koole W, van Schendel R, Karambelas AE, van Heteren JT, Okihara KL, Tijsterman M. A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nature communications. 2014;5:3216. Epub 2014/02/06. doi: 10.1038/ncomms4216 24496117.

31. Sarkies P, Reams C, Simpson LJ, Sale JE. Epigenetic instability due to defective replication of structured DNA. Molecular cell. 2010;40(5):703–13. Epub 2010/12/15. doi: 10.1016/j.molcel.2010.11.009 21145480; PubMed Central PMCID: PMC3145961.

32. Schiavone D, Guilbaud G, Murat P, Papadopoulou C, Sarkies P, Prioleau MN, et al. Determinants of G quadruplex-induced epigenetic instability in REV1-deficient cells. The EMBO journal. 2014;33(21):2507–20. Epub 2014/09/06. doi: 10.15252/embj.201488398 25190518; PubMed Central PMCID: PMC4282387.

33. van Schendel R, Roerink SF, Portegijs V, van den Heuvel S, Tijsterman M. Polymerase Θ is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nature communications. 2015;6:7394. Epub 2015/06/17. doi: 10.1038/ncomms8394 26077599; PubMed Central PMCID: PMC4490562.

34. van Schendel R, van Heteren J, Welten R, Tijsterman M. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining. PLoS genetics. 2016;12(10):e1006368. Epub 2016/10/19. doi: 10.1371/journal.pgen.1006368 27755535; PubMed Central PMCID: PMC5068794.

35. Shao Z, Niwa S, Higashitani A, Daigaku Y. Vital roles of PCNA K165 modification during C. elegans gametogenesis and embryogenesis. DNA repair. 2019;82:102688. Epub 2019/08/27. doi: 10.1016/j.dnarep.2019.102688 31450086.

36. Nelson JR, Lawrence CW, Hinkle DC. Deoxycytidyl transferase activity of yeast REV1 protein. Nature. 1996;382(6593):729–31. Epub 1996/08/22. doi: 10.1038/382729a0 8751446.

37. Haracska L, Unk I, Johnson RE, Johansson E, Burgers PM, Prakash S, et al. Roles of yeast DNA polymerases delta and zeta and of Rev1 in the bypass of abasic sites. Genes & development. 2001;15(8):945–54. Epub 2001/04/24. doi: 10.1101/gad.882301 11316789; PubMed Central PMCID: PMC312678.

38. Zhang Y, Wu X, Rechkoblit O, Geacintov NE, Taylor JS, Wang Z. Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion. Nucleic acids research. 2002;30(7):1630–8. Epub 2002/03/28. doi: 10.1093/nar/30.7.1630 11917024; PubMed Central PMCID: PMC101843.

39. Zhou Y, Wang J, Zhang Y, Wang Z. The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis. Nucleic acids research. 2010;38(15):5036–46. Epub 2010/04/15. doi: 10.1093/nar/gkq225 20388628; PubMed Central PMCID: PMC2926598.

40. 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. The Journal of biological chemistry. 2012;287(40):33847–52. Epub 2012/08/04. doi: 10.1074/jbc.M112.396838 22859296; PubMed Central PMCID: PMC3460479.

41. 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. The Journal of biological chemistry. 2012;287(31):26400–8. Epub 2012/06/16. doi: 10.1074/jbc.M112.380998 22700975; PubMed Central PMCID: PMC3406723.

42. 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. The Journal of biological chemistry. 2012;287(40):33836–46. Epub 2012/08/04. doi: 10.1074/jbc.M112.394841 22859295; PubMed Central PMCID: PMC3460478.

43. Waters LS, Minesinger BK, Wiltrout ME, D'Souza S, Woodruff RV, Walker GC. Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance. Microbiology and molecular biology reviews: MMBR. 2009;73(1):134–54. Epub 2009/03/05. doi: 10.1128/MMBR.00034-08 19258535; PubMed Central PMCID: PMC2650891.

44. Fuchs RP. Tolerance of lesions in E. coli: Chronological competition between Translesion Synthesis and Damage Avoidance. DNA repair. 2016;44:51–8. Epub 2016/06/21. doi: 10.1016/j.dnarep.2016.05.006 27321147.

45. Unk I, Hajdú I, Blastyák A, Haracska L. Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance. DNA repair. 2010;9(3):257–67. Epub 2010/01/26. doi: 10.1016/j.dnarep.2009.12.013 20096653.

46. Jansen JG, Tsaalbi-Shtylik A, Hendriks G, Gali H, Hendel A, Johansson F, et al. Separate domains of Rev1 mediate two modes of DNA damage bypass in mammalian cells. Molecular and cellular biology. 2009;29(11):3113–23. Epub 2009/04/01. doi: 10.1128/MCB.00071-09 19332561; PubMed Central PMCID: PMC2682010.

47. Kosarek JN, Woodruff RV, Rivera-Begeman A, Guo C, D'Souza S, Koonin EV, et al. Comparative analysis of in vivo interactions between Rev1 protein and other Y-family DNA polymerases in animals and yeasts. DNA repair. 2008;7(3):439–51. Epub 2008/02/05. doi: 10.1016/j.dnarep.2007.11.016 18242152; PubMed Central PMCID: PMC2363158.

48. Jansen JG, Langerak P, Tsaalbi-Shtylik A, van den Berk P, Jacobs H, de Wind N. Strand-biased defect in C/G transversions in hypermutating immunoglobulin genes in Rev1-deficient mice. The Journal of experimental medicine. 2006;203(2):319–23. Epub 2006/02/16. doi: 10.1084/jem.20052227 16476771; PubMed Central PMCID: PMC2118202.

49. Tomas-Roca L, Tsaalbi-Shtylik A, Jansen JG, Singh MK, Epstein JA, Altunoglu U, et al. De novo mutations in PLXND1 and REV3L cause Möbius syndrome. Nature communications. 2015;6:7199. Epub 2015/06/13. doi: 10.1038/ncomms8199 26068067; PubMed Central PMCID: PMC4648025.

50. Bemark M, Khamlichi AA, Davies SL, Neuberger MS. Disruption of mouse polymerase zeta (Rev3) leads to embryonic lethality and impairs blastocyst development in vitro. Current biology: CB. 2000;10(19):1213–6. Epub 2000/10/26. doi: 10.1016/s0960-9822(00)00724-7 11050391.

51. Lemmens BB, Tijsterman M. DNA double-strand break repair in Caenorhabditis elegans. Chromosoma. 2011;120(1):1–21. Epub 2010/11/06. doi: 10.1007/s00412-010-0296-3 21052706; PubMed Central PMCID: PMC3028100.

52. Boiteux S, Coste F, Castaing B. Repair of 8-oxo-7,8-dihydroguanine in prokaryotic and eukaryotic cells: Properties and biological roles of the Fpg and OGG1 DNA N-glycosylases. Free radical biology & medicine. 2017;107:179–201. Epub 2016/12/03. doi: 10.1016/j.freeradbiomed.2016.11.042 27903453.

53. Haracska L, Yu SL, Johnson RE, Prakash L, Prakash S. Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase eta. Nature genetics. 2000;25(4):458–61. Epub 2000/08/10. doi: 10.1038/78169 10932195.

54. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71–94. Epub 1974/05/01. 4366476; PubMed Central PMCID: PMC1213120.

55. Cuppen E, Gort E, Hazendonk E, Mudde J, van de Belt J, Nijman IJ, et al. Efficient target-selected mutagenesis in Caenorhabditis elegans: toward a knockout for every gene. Genome research. 2007;17(5):649–58. Epub 2007/04/10. doi: 10.1101/gr.6080607 17416746; PubMed Central PMCID: PMC1855173.

56. Waaijers S, Portegijs V, Kerver J, Lemmens BB, Tijsterman M, van den Heuvel S, et al. CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans. Genetics. 2013;195(3):1187–91. Epub 2013/08/28. doi: 10.1534/genetics.113.156299 23979586; PubMed Central PMCID: PMC3813849.

57. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics (Oxford, England). 2009;25(14):1754–60. Epub 2009/05/20. doi: 10.1093/bioinformatics/btp324 19451168; PubMed Central PMCID: PMC2705234.

58. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature genetics. 2011;43(5):491–8. Epub 2011/04/12. doi: 10.1038/ng.806 21478889; PubMed Central PMCID: PMC3083463.

59. Ye K, Schulz MH, Long Q, Apweiler R, Ning Z. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics (Oxford, England). 2009;25(21):2865–71. Epub 2009/06/30. doi: 10.1093/bioinformatics/btp394 19561018; PubMed Central PMCID: PMC2781750.

60. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nature biotechnology. 2011;29(1):24–6. Epub 2011/01/12. doi: 10.1038/nbt.1754 21221095; PubMed Central PMCID: PMC3346182.

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