title: Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology.
abstract: The defining event in homologous recombination is the exchange of base-paired partners between a single-stranded (ss) DNA and a homologous duplex driven by recombinase proteins, such as human RAD51. To understand the mechanism of this essential genome maintenance event, we analyzed the structure of RAD51-DNA complexes representing strand exchange intermediates at nanometer resolution by scanning force microscopy. Joint molecules were formed between substrates with a defined ssDNA segment and homologous region on a double-stranded (ds) partner. We discovered and quantified several notable architectural features of RAD51 joint molecules. Each end of the RAD51-bound joints had a distinct structure. Using linear substrates, a 10-nt region of mispaired bases blocked extension of joint molecules in all examples observed, whereas 4 nt of heterology only partially blocked joint molecule extension. Joint molecules, including 10 nt of heterology, had paired DNA on either side of the heterologous substitution, indicating that pairing could initiate from the free 3'end of ssDNA or from a region adjacent to the ss-ds junction. RAD51 filaments covering joint ss-dsDNA regions were more stable to disassembly than filaments covering dsDNA. We discuss how distinct structural features of RAD51-bound DNA joints can play important roles as recognition sites for proteins that facilitate and control strand exchange.
authors.affiliation: Department of Cell Biology and Genetics, Cancer Genomics Center, Erasmus MC, CA Rotterdam, The Netherlands.
authors.forename: D
authors.initials: D
authors.lastname: Ristic
authors.forename: R
authors.initials: R
authors.lastname: Kanaar
authors.forename: C
authors.initials: C
authors.lastname: Wyman
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D006801:Humans; D018625:Microscopy, Atomic Force; D051135:Rad51 Recombinase / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure*; D011995:Recombination, Genetic
chemical_list: D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase
references.citation: Nucleic Acids Res. 2001 Jun 15;29(12):2464-70
references.pmid: 11410652
references.citation: Mol Cell. 2008 Feb 29;29(4):510-6
references.pmid: 18313388
references.citation: Annu Rev Genet. 2006;40:363-83
references.pmid: 16895466
references.citation: Mol Cell. 2007 Nov 9;28(3):482-90
references.pmid: 17996711
references.citation: Mol Cell. 2007 Nov 9;28(3):468-81
references.pmid: 17996710
references.citation: Mol Cell. 2005 Dec 9;20(5):783-92
references.pmid: 16337601
references.citation: Nucleic Acids Res. 2005 Jun 08;33(10):3292-302
references.pmid: 15944450
references.citation: J Biol Chem. 2006 Sep 8;281(36):26268-79
references.pmid: 16831867
references.citation: Exp Cell Res. 2006 Aug 15;312(14):2660-5
references.pmid: 16859683
references.citation: Genes Dev. 2009 Jan 1;23(1):67-79
references.pmid: 19136626
references.citation: Nat Struct Mol Biol. 2004 Aug;11(8):791-6
references.pmid: 15235592
references.citation: Science. 1993 Mar 26;259(5103):1896-9
references.pmid: 8456314
references.citation: Nucleic Acids Res. 2007;35(21):7171-87
references.pmid: 17947332
references.citation: Annu Rev Biochem. 2008;77:229-57
references.pmid: 18275380
references.citation: Nucleic Acids Res. 2001 Dec 15;29(24):5052-7
references.pmid: 11812836
references.citation: Curr Protein Pept Sci. 2004 Apr;5(2):73-9
references.pmid: 15078218
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3077-82
references.pmid: 19204284
references.citation: J Mol Biol. 2004 Apr 2;337(4):817-27
references.pmid: 15033353
references.citation: Structure. 2003 Feb;11(2):187-96
references.pmid: 12575938
references.citation: Cold Spring Harb Symp Quant Biol. 1984;49:553-9
references.pmid: 6397310
references.citation: Proc Natl Acad Sci U S A. 2009 Jan 13;106(2):361-8
references.pmid: 19122145
references.citation: Nature. 2000 Mar 2;404(6773):37-41
references.pmid: 10716434
references.citation: Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8419-24
references.pmid: 11459984
references.citation: J Biol Chem. 2009 Mar 20;284(12):7505-17
references.pmid: 19150983
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 6;101(27):9988-93
references.pmid: 15226506
references.citation: Nucleic Acids Res. 2006 May 02;34(8):2269-79
references.pmid: 16670433
references.citation: Structure. 2007 May;15(5):599-609
references.pmid: 17502105
references.citation: Nature. 1982 Sep 9;299(5879):185-6
references.pmid: 7050731
references.citation: Nature. 2008 May 22;453(7194):489-4
references.pmid: 18497818
references.citation: EMBO J. 1994 Dec 1;13(23):5764-71
references.pmid: 7988572
references.citation: Nucleic Acids Res. 2007;35(12):4124-40
references.pmid: 17567608
references.citation: J Biol Chem. 1987 Mar 25;262(9):4011-6
references.pmid: 2951381
references.citation: Nucleic Acids Res. 2007;35(17):5646-57
references.pmid: 17709342
references.citation: Mol Cell Biol. 2006 Feb;26(3):976-89
references.pmid: 16428451
references.citation: Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7618-22
references.pmid: 8755524
references.citation: Nature. 2009 Feb 5;457(7230):745-8
references.pmid: 19060884
references.citation: Nat Struct Mol Biol. 2007 Aug;14(8):746-53
references.pmid: 17660833
references.citation: J Biol Chem. 1985 Jan 10;260(1):165-9
references.pmid: 2981202
references.citation: Nucleic Acids Res. 2009 Feb;37(2):638-46
references.pmid: 19074197