title: Coincident resection at both ends of random, γ-induced double-strand breaks requires MRX (MRN), Sae2 (Ctp1), and Mre11-nuclease.
abstract: Resection is an early step in homology-directed recombinational repair (HDRR) of DNA double-strand breaks (DSBs). Resection enables strand invasion as well as reannealing following DNA synthesis across a DSB to assure efficient HDRR. While resection of only one end could result in genome instability, it has not been feasible to address events at both ends of a DSB, or to distinguish 1- versus 2-end resections at random, radiation-induced "dirty" DSBs or even enzyme-induced "clean" DSBs. Previously, we quantitatively addressed resection and the role of Mre11/Rad50/Xrs2 complex (MRX) at random DSBs in circular chromosomes within budding yeast based on reduced pulsed-field gel electrophoretic mobility ("PFGE-shift"). Here, we extend PFGE analysis to a second dimension and demonstrate unique patterns associated with 0-, 1-, and 2-end resections at DSBs, providing opportunities to examine coincidence of resection. In G2-arrested WT, Δrad51 and Δrad52 cells deficient in late stages of HDRR, resection occurs at both ends of γ-DSBs. However, for radiation-induced and I-SceI-induced DSBs, 1-end resections predominate in MRX (MRN) null mutants with or without Ku70. Surprisingly, Sae2 (Ctp1/CtIP) and Mre11 nuclease-deficient mutants have similar responses, although there is less impact on repair. Thus, we provide direct molecular characterization of coincident resection at random, radiation-induced DSBs and show that rapid and coincident initiation of resection at γ-DSBs requires MRX, Sae2 protein, and Mre11 nuclease. Structural features of MRX complex are consistent with coincident resection being due to an ability to interact with both DSB ends to directly coordinate resection. Interestingly, coincident resection at clean I-SceI-induced breaks is much less dependent on Mre11 nuclease or Sae2, contrary to a strong dependence on MRX complex, suggesting different roles for these functions at "dirty" and clean DSB ends. These approaches apply to resection at other DSBs. Given evolutionary conservation, the observations are relevant to DNA repair in human cells.
authors.affiliation: Chromosome Stability Section, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.
authors.forename: James W
authors.initials: JW
authors.lastname: Westmoreland
authors.forename: Michael A
authors.initials: MA
authors.lastname: Resnick
mesh_terms: D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004261:DNA Replication; D016521:Electrophoresis, Gel, Pulsed-Field; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005720:Gamma Rays; D042822:Genomic Instability / Q000528:radiation effects; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D059767:Recombinational DNA Repair / Q000235:genetics* / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; C107849:SAE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae
references.citation: Cell. 2001 Jul 13;106(1):59-70
references.pmid: 11461702
references.citation: J Biol Chem. 2011 Apr 15;286(15):13669-80
references.pmid: 21335604
references.citation: Annu Rev Genet. 2011;45:247-71
references.pmid: 21910633
references.citation: Trends Biochem Sci. 2009 May;34(5):264-72
references.pmid: 19375328
references.citation: Curr Biol. 2004 Dec 14;14(23):2107-12
references.pmid: 15589152
references.citation: DNA Repair (Amst). 2003 Mar 1;2(3):243-58
references.pmid: 12547388
references.citation: J Biol Chem. 2005 Nov 18;280(46):38631-8
references.pmid: 16162495
references.citation: EMBO J. 2010 Oct 6;29(19):3370-80
references.pmid: 20834227
references.citation: DNA Repair (Amst). 2005 May 2;4(5):606-17
references.pmid: 15811632
references.citation: Mol Cell Biol. 2011 Nov;31(21):4379-89
references.pmid: 21876003
references.citation: Cell. 2010 Apr 16;141(2):243-54
references.pmid: 20362325
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11845-50
references.pmid: 18701715
references.citation: PLoS Genet. 2009 Sep;5(9):e1000656
references.pmid: 19763170
references.citation: Mol Cell Biol. 1999 Jan;19(1):556-66
references.pmid: 9858579
references.citation: Nucleic Acids Res. 2008 Apr;36(6):1836-46
references.pmid: 18267974
references.citation: Mol Cell Biol. 2004 Nov;24(21):9682-94
references.pmid: 15485933
references.citation: Methods Enzymol. 2006;409:329-45
references.pmid: 16793410
references.citation: Nat Genet. 1999 Sep;23(1):81-5
references.pmid: 10471504
references.citation: Nat Struct Mol Biol. 2010 Dec;17(12):1478-85
references.pmid: 21102445
references.citation: J Theor Biol. 1976 Jun;59(1):97-106
references.pmid: 940351
references.citation: Nature. 2011 Oct 16;479(7372):241-4
references.pmid: 22002605
references.citation: Mol Cell. 2007 Nov 30;28(4):638-51
references.pmid: 18042458
references.citation: Curr Biol. 2004 Dec 14;14(23):2096-106
references.pmid: 15589151
references.citation: J Biol Chem. 2011 Dec 30;286(52):44945-51
references.pmid: 22039049
references.citation: Nature. 2010 Sep 2;467(7311):108-11
references.pmid: 20811460
references.citation: Nat Biotechnol. 2001 Aug;19(8):773-6
references.pmid: 11479573
references.citation: DNA Repair (Amst). 2010 Aug 5;9(8):914-21
references.pmid: 20663718
references.citation: Genes Dev. 2009 Feb 1;23(3):291-303
references.pmid: 19204116
references.citation: DNA Repair (Amst). 2010 Dec 10;9(12):1283-91
references.pmid: 21050828
references.citation: Mol Cell. 2001 Nov;8(5):1129-35
references.pmid: 11741547
references.citation: Cell. 2008 Oct 3;135(1):97-109
references.pmid: 18854158
references.citation: PLoS Genet. 2011 Sep;7(9):e1002271
references.pmid: 21931565
references.citation: DNA Repair (Amst). 2011 Jan 2;10(1):102-10
references.pmid: 21115410
references.citation: Cell. 2008 Oct 3;135(1):85-96
references.pmid: 18854157
references.citation: DNA Repair (Amst). 2011 Mar 7;10(3):344-8
references.pmid: 21227759
references.citation: Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6590-5
references.pmid: 22493268
references.citation: Genetics. 2001 May;158(1):109-22
references.pmid: 11333222
references.citation: Cell. 2004 Sep 17;118(6):699-713
references.pmid: 15369670
references.citation: EMBO J. 2010 Oct 6;29(19):3358-69
references.pmid: 20729809
references.citation: EMBO J. 2010 Sep 1;29(17):2864-74
references.pmid: 20647996
references.citation: Genetics. 1996 Jun;143(2):741-54
references.pmid: 8725223
references.citation: EMBO J. 1993 Jul;12(7):2939-47
references.pmid: 8335007
references.citation: Nature. 2008 Oct 2;455(7213):689-92
references.pmid: 18716619
references.citation: Yeast. 1999 Oct;15(14):1541-53
references.pmid: 10514571
references.citation: Cell. 2008 Sep 19;134(6):981-94
references.pmid: 18805091
references.citation: Nat Rev Mol Cell Biol. 2011 Feb;12(2):90-103
references.pmid: 21252998
references.citation: PLoS Genet. 2008 Nov;4(11):e1000264
references.pmid: 19023402
references.citation: PLoS Genet. 2011 Apr;7(4):e1002059
references.pmid: 21552545
references.citation: Genetics. 2005 Dec;171(4):1561-70
references.pmid: 16143598
references.citation: Nat Struct Mol Biol. 2010 Jan;17(1):11-6
references.pmid: 20051983
references.citation: Nature. 2008 Oct 9;455(7214):770-4
references.pmid: 18806779
references.citation: Mol Cell Biol. 2005 Feb;25(3):933-44
references.pmid: 15657422