DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2.
The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3'-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5'-terminated strand of the DNA break and to inhibit 3' to 5' degradation by Dna2, actions that generate and protect the 3'-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11-Rad50-Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3-Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3-Rmi1 and MRX are important for recruitment of the Sgs1-Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes.
Cejka
Petr
P
Department of Microbiology, University of California, Davis, Davis, California 95616-8665, USA.
Cannavo
Elda
E
Polaczek
Piotr
P
Masuda-Sasa
Taro
T
Pokharel
Subhash
S
Campbell
Judith L
JL
Kowalczykowski
Stephen C
SC
D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D053484:RecQ Helicases / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; C086930:RAD50 protein, S cerevisiae; C082728:RFA1 protein, S cerevisiae; D051716:Replication Protein A; C502206:Rmi1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C502374:TOP3 protein, S cerevisiae; C086929:XRS2 protein, S cerevisiae; D003851:Deoxyribonucleases; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae; D053484:RecQ Helicases
Nature. 2001 Jul 26;412(6845):456-61
11473323
Mol Cell Biol. 2004 Nov;24(21):9682-94
15485933
Cell. 1997 Jul 11;90(1):77-86
9230304
J Biol Chem. 2010 Mar 12;285(11):8290-301
20086270
EMBO J. 2005 Jun 1;24(11):2024-33
15889139
Proc Natl Acad Sci U S A. 2008 Nov 4;105(44):16906-11
18971343
J Biol Chem. 2004 Apr 9;279(15):15014-24
14747468
Mol Cell Biol. 1994 Dec;14(12):8391-8
7969174
Mol Cell. 2009 Jan 16;33(1):117-23
19150433
Genes Dev. 2009 May 15;23(10):1234-45
19451222
J Biol Chem. 2001 Jun 1;276(22):19375-81
11278509
Mol Cell Biol. 2005 Jul;25(13):5738-51
15964827
Mol Cell. 2009 Nov 25;36(4):704-13
19941829
J Biol Chem. 1995 Nov 10;270(45):26766-9
7592912
Science. 1995 Feb 24;267(5201):1166-9
7855597
EMBO J. 2003 Aug 15;22(16):4325-36
12912929
Nature. 2005 Aug 18;436(7053):1053-7
16107854
Annu Rev Genet. 2004;38:233-71
15568977
J Mol Biol. 2001 Apr 13;307(5):1207-21
11292336
Genes Dev. 2008 Oct 15;22(20):2767-72
18923075
Microbiol Mol Biol Rev. 2008 Dec;72(4):642-71, Table of Contents
19052323
DNA Repair (Amst). 2004 Aug-Sep;3(8-9):845-54
15279769
Nucleic Acids Res. 2003 Jun 15;31(12):3006-15
12799426
EMBO J. 2008 Jul 9;27(13):1875-85
18511906
J Biol Chem. 2008 Sep 5;283(36):24359-73
18593712
Cell. 2008 Sep 19;134(6):981-94
18805091
J Biol Chem. 2000 Dec 1;275(48):38022-31
10984490
Genes Dev. 1998 Apr 15;12(8):1134-44
9553043
PLoS One. 2009;4(1):e4267
19165339
Mol Cell Biol. 2005 Jun;25(11):4476-87
15899853
J Biol Chem. 2000 Jun 2;275(22):16518-29
10748138
Nature. 2008 Oct 9;455(7214):770-4
18806779
J Mol Biol. 1992 Sep 5;227(1):54-71
1522601