DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr540 amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.
Vyšlo v časopise: Yeast Pol4 Promotes Tel1-Regulated Chromosomal Translocations. PLoS Genet 9(7): e32767. doi:10.1371/journal.pgen.1003656 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003656
1. PardoB, Gómez-GonzálezB, AguileraA (2009) DNA repair in mammalian cells: DNA double-strand break repair: how to fix a broken relationship. Cell Mol Life Sci 66 : 1039–1056.
2. YuX, GabrielA (2004) Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining. Genetics 166 : 741–751.
3. LieberMR, GuJ, LuH, ShimazakiN, TsaiAG (2010) Nonhomologous DNA End Joining (NHEJ) and Chromosomal Translocations in Humans. H.P. Nasheuer (ed.) Genome Stability and Human Diseases, Subcellular Biochemistry 50 : 279–296.
4. ElliottB, RichardsonC, JasinM (2005) Chromosomal translocation mechanisms at intronic alu elements in mammalian cells. Mol Cell 17 : 885–94.
5. NussenzweigA, NussenzweigMC (2010) Origin of chromosomal translocations in lymphoid cancer. Cell 141 : 27–38.
6. GostissaM, AltFW, ChiarleR (2011) Mechanisms that Promote and Suppress Chromosomal Translocations in Lymphocytes. Annu Rev Biochem 29 : 319–350.
7. LieberMR (2010) The mechanism of double-strand break DNA repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79 : 181–211.
8. DaleyJM, PalmbosPL, WuD, WilsonTE (2005) Nonhomologous end joining in yeast. Annu Rev Genet 39 : 431–451.
9. McVeyM, LeeSE (2008) MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. Trends Genet 24 : 529–538.
10. RamsdenDA, AsagoshiK (2012) DNA Polymerases in Nonhomologous End Joining: Are There Any Benefits to Standing Out from the Crowd. Environmental and Molecular Mutagenesis 53 : 741–751.
11. AkopiantsK, ZhouR-Z, MohapatraS, ValerieK, Lees-MillerS, et al. (2009) Requirement for XLF/Cernunnos in alignment based gap filling by DNA polymerases λ and μ for nonhomologous end joining in human whole-cell extracts. Nucleic Acids Res 37 : 4055–4062.
12. MoonAF, Garcia-DiazM, BatraVK, BeardWA, BebenekK, et al. (2007) The X family portrait: structural insights into biological functions of X family polymerases. DNA Repair (Amst) 6 : 1709–1725.
13. MartinMJ, JuárezR, BlancoL (2012) DNA binding determinants promoting NHEJ by human Polμ. Nucleic Acids Res 40 : 11389–11403.
14. ZhangY, WuX, YuanF, XieZ, WangZ (2001) Highly frequent frameshift DNA synthesis by human DNA polymerase mu. Mol Cell Biol 21 : 7995–8006.
15. RuizJF, LucasD, García-PalomeroE, SaezAI, GonzálezMA, et al. (2004) Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate. Nucleic Acids Res 32 : 5861–5873.
16. PicherAJ, García-DíazM, BebenekK, PedersenLC, KunkelTA, et al. (2006) Promiscuous mismatch extension by human DNA polymerase lambda. Nucleic Acids Res 34 : 3259–3266.
17. MahajanKN, Nick McElhinnySA, MitchellBS, RamsdenDA (2002) Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair. Mol Cell Biol 22 : 5194–5202.
18. MaY, LuH, TippinB, GoodmanMF, ShimazakiN, et al. (2005) A Biochemically Defined System for Mammalian Nonhomologous DNA End Joining. Molecular Cell 16 : 701–713.
19. Nick McElhinnySA, HavenerJM, Garcia-DiazM, JuarezR, BebenekK, et al. (2005) A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. Mol Cell 19 : 357–366.
20. GuJ, LuH, TippinB, ShimazakiN, GoodmanMF, LieberMR (2007) XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps. The EMBO Journal 26 : 1010–1023.
21. CappJP, BoudsocqF, BertrandP, Laroche-ClaryA, PourquierP, LopezBS, CazauxC, HoffmannJS, CanitrotY (2006) The DNA polymerase lambda is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells. Nucleic Acids Res 34 : 2998–3007.
22. CappJP, BoudsocqF, BesnardA-G, LopezBS, CazauxC, et al. (2007) Involvement of DNA polymerase μ in the repair of a specific subset of DNA double-strand breaks in mammalian cells. Nucleic Acids Res 35 : 3551–3560.
23. OgiwaraH, KohnoT (2011) Essential factors for incompatible DNA end joining at chromosomal DNA double strand breaks in vivo. PLoS One 6 (12) e28756.
24. ChayotR, MontagneB, RicchettiM (2012) DNA polymerase μ is a global player in the repair of non-homologous end-joining substrates. DNA Repair (Amst) 11 : 22–34.
25. González-BarreraS, SanchezA, RuizJF, JuarezR, PicherA, et al. (2005) Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe. Nucleic Acids Res 33 : 4762–4774.
26. BebenekK, Garcia-DiazM, PatishallSR, KunkelTA (2005) Biochemical Properties of Saccharomyces cerevisiae DNA Polymerase IV. J Biol Chem 280 : 20051–20058.
27. TsengHM, TomkinsonAE (2002) A physical and functional interaction between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in nonhomologous end joining. J Biol Chem 277 : 45630–45637.
28. WilsonTE, LieberMR (1999) Efficient processing of DNA ends during yeast nonhomologous end joining. Evidence for a DNA polymerase beta (Pol4)-dependent pathway. J Biol Chem 274 : 23599–609.
29. PardoB, MaE, MarcandS (2006) Mismatch Tolerance by DNA Polymerase Pol4 in the Course of Nonhomologous End Joining in Saccharomyces cerevisiae. Genetics 172 : 2689–2694.
30. DaleyJM, LaanRL, SureshA, WilsonTE (2005) DNA joint dependence of pol X family polymerase action in nonhomologous end joining. J Biol Chem 280 : 29030–29037.
31. DaleyJM, WilsonTE (2008) Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining. DNA Repair (Amst) 7 : 67–76.
32. LeeK, LeeSE (2007) Saccharomyces cerevisiae Sae2 - and Tel1-Dependent Single-Strand DNA Formation at DNA Break Promotes Microhomology-Mediated End Joining. Genetics 176 : 2003–2014.
33. TsengS-F, GabrielA, TengS-C (2008) Proofreading Activity of DNA Polymerase Pol2 Mediates 3′-End Processing during Nonhomologous End Joining in Yeast. PLoS Genet 4 (4) e1000060.
34. MarcandS, PardoB, GratiasA, CahunS, CallebautI (2008) Multiple pathways inhibit NHEJ at telomeres. Genes Dev 22 : 1153–1158.
35. RuizJF, Gómez-GonzálezB, AguileraA (2011) AID induces double-strand breaks at immunoglobulin switch regions and c-MYC causing chromosomal translocations in yeast THO mutants. PLoS Genet 7 (2) e1002009.
36. MooreJK, HaberJE (1996) Cell Cycle and Genetic Requirements of Two Pathways of Nonhomologous End-Joining Repair of Double-Strand Breaks in Saccharomyces cerevisiae. Mol Cell Biol 16 : 2164–2173.
37. DavisBJ, HavenerJM, RamsdenDA (2008) End-bridging is required for polμ to efficiently promote repair of noncomplementary ends by nonhomologous end joining. Nucleic Acids Res 36 : 3085–3094.
38. LeeK, ZhangY, LeeSE (2008) Saccharomyces cerevisiae ATM orthologue suppresses break-induced chromosome translocations. Nature 454 : 543–547.
39. BennardoN, StarkJM (2010) ATM Limits Incorrect End Utilization during Non-Homologous End Joining of Multiple Chromosome Breaks. PLoS Genet 6: e1001194.
40. MajoryJC, PetesTD (2000) Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase. Proc Natl Acad Sci USA 97 : 13749–13754.
41. ChanCY, GalliA, SchiestlRH (2008) Pol3 is involved in nonhomologous end-joining in Saccharomyces cerevisiae. DNA Repair (Amst) 7 : 1531–1541.
42. BertocciB, De SmetA, BerekC, WeillJ-C, ReynaudC-A (2003) Immunoglobulin Light Chain Gene Rearrangement Is Impaired in Mice Deficient for DNA Polymerase Mu. Immunity 19 : 203–211.
43. LieberMR (2008) Flexibility in the order of action and in the enzymology of the nuclease, polymerases, and ligase of vertebrate nonhomologous DNA end joining: relevance to cancer, aging, and the immune system. Cell Res 18 : 125–133.
44. PoloSE, JacksonSP (2011) Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev 25 : 409–433.
45. McCulleyJL, PetesTD (2010) Chromosome rearrangements and aneuploidy in yeast strains lacking both Tel1p and Mec1p reflect deficiencies in two different mechanisms. Proc Natl Acad Sci USA 107 : 11465–11470.
46. MyungK, DattaA, KolodnerRD (2001) Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Cell 104 : 397–40.
47. MatsuokaS, BallifBA, SmogorzewskaA, McDonaldER, HurovKE, et al. (2007) ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage. Science 316 : 1160–1166.
48. Garcia-DiazM, BebenekK, KrahnJM, KunkelTA, PedersenLC (2005) A closed conformation for the Pol lambda catalytic cycle. Nat Struct Mol Biol 12 : 97–98.
49. MisteliT, SoutoglouE (2009) The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol 10 : 243–254.
50. SimsekD, JasinM (2010) Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation. Nat Struct Mol Biol 17 : 410–416.
51. LamHYK, MuXJ, StutzAM, TanzerA, CaytingPD, et al. (2010) Nucleotide-resolution analysis of structural variants using BreakSeq and a breakpoint library. Nat Biotech 28 : 47–55.
52. RuizJF, Gómez-GonzálezB, AguileraA (2009) Chromosomal translocations caused by either pol32-dependent or pol32-independent triparental break-induced replication. Mol Cell Biol 29 : 5441–54.
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