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Mismatch Repair Inhibits Homeologous Recombination Via Coordinated Directional Unwinding of Trapped DNA Structures

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Mismatch Repair Inhibits Homeologous Recombination Via Coordinated Directional Unwinding of Trapped DNA Structures Molecular Cell Article Mismatch Repair Inhibits Homeologous Recombination via Coordinated Directional Unwinding of Trapped DNA Structures Khek-Chian Tham,1 Nicolaas Hermans,1 Herrie H.K. Winterwerp,3 Michael M. Cox,4 Claire Wyman,1,2 Roland Kanaar,1,2 and Joyce H.G. Lebbink1,2,* 1Department of Genetics, Cancer Genomics Netherlands 2Department of Radiation Oncology Erasmus Medical Center, Rotterdam 3000 CA, The Netherlands 3Division of Biochemistry and Center for Biomedical Genetics, Netherlands Cancer Institute, Amsterdam 1066 CX, The Netherlands 4Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.07.008 SUMMARY transduction (Zahrt and Maloy, 1997), transformation (Majewski et al., 2000), and conjugational E. coli-E. coli crosses that involve Homeologous recombination between divergent the creation of mismatches (Feinstein and Low, 1986). Likewise, DNA sequences is inhibited by DNA mismatch repair. in eukaryotes, individual MMR proteins have roles of varying In Escherichia coli, MutS and MutL respond to DNA magnitude in the prevention of homeologous recombination mismatches within recombination intermediates (Selva et al., 1995; Datta et al., 1996; Nicholson et al., 2000; de and prevent strand exchange via an unknown mech- Wind et al., 1995). Inhibition of homeologous reactions is thought anism. Here, using purified proteins and DNA sub- to protect the genome from recombination events between divergent repeats (de Wind et al., 1999). This is important in hu- strates, we find that in addition to mismatches within mans, in which the amount of repetitive DNA is as high as 50%. the heteroduplex region, secondary structures within An intriguing question is how MMR and recombination path- the displaced single-stranded DNA formed during ways are integrated at the molecular level. Common events dur- branch migration within the recombination interme- ing MMR and antirecombination include MutS binding to a DNA diate are involved in the inhibition. We present a mismatch, complex formation between MutS and MutL, and model that explains how higher-order complex for- MutL-mediated orchestration of MutH (GATC endonuclease) mation of MutS, MutL, and DNA blocks branch and UvrD (30-50 helicase) (Jiricny 2013; Stambuk and Radman, migration by preventing rotation of the DNA strands 1998). MutS and MutL are potent suppressors of homeologous within the recombination intermediate. Furthermore, recombination in vivo (Rayssiguier et al., 1989). Biochemical we find that the helicase UvrD is recruited to direc- studies demonstrate that MutS and MutL block RecA-mediated tionally resolve these trapped intermediates toward homeologous strand exchange through prevention of branch migration (Worth et al., 1994). However, the molecular mecha- DNA substrates. Thus, our results explain on a mech- nism of this inhibition remains unknown. DNA mismatches do anistic level how the coordinated action between not occur within recombination filaments until after strand ex- MutS, MutL, and UvrD prevents homeologous change has taken place, and somehow MMR proteins are able recombination and maintains genome stability. to inhibit the ongoing branch migration into a region where mis- matches are not yet present. Although the fate of the trapped homeologous strand-exchange intermediates is unknown, cells INTRODUCTION must disassemble these problematic DNA structures. Initially, genetic data suggested a minor role for the helicase UvrD in DNA mismatch repair (MMR) is one of several important DNA this process (Rayssiguier et al., 1989). However, the homeolo- repair processes conserved from bacteria to mammals. MMR re- gous recombination frequency in a recipient strain lacking both pairs base-base mismatches and insertion/deletion loops gener- MutH and UvrD is as high as in mutS and mutL strains (Stambuk ated during replication (Jiricny, 2013). In addition, MMR proteins and Radman, 1998). Based on this, the fate of MutS-MutL- prevent illegitimate recombination between divergent (homeolo- trapped recombination intermediates was hypothesized to gous) sequences. Thus, inactivation of MMR genes not only re- depend on two distinct mechanisms. One is MutH-independent sults in mutator but also in hyperrecombination phenotypes. and involves MutS, MutL, and UvrD during the early stage of The increased frequency (up to 1,000-fold) of interspecies conju- homeologous recombination. The binding of a mismatch by gation between Escherichia coli and Salmonella typhimurium in the MutS-MutL complex is proposed to recruit UvrD to the near- mutS, mutL, mutH, and uvrD recipients indicates that MMR est single-strand/double-strand DNA (ssDNA/dsDNA) junction acts as a barrier to homeologous recombination (Rayssiguier of the recombination intermediate. UvrD then unwinds the het- et al., 1989, 1991). Similar findings are obtained for interspecies eroduplex from the loading junction toward the mismatch and 326 Molecular Cell 51, 326–337, August 8, 2013 ª2013 Elsevier Inc. Molecular Cell Antirecombination Mechanism of DNA Mismatch Repair Figure 1. MutS-MutL Inhibits Progression A Homologous Homeologous B Branched Intermediate 5 3.0 nm of DNA Strand Exchange during Homeolo- 0 15 30 45 60 0 15 30 45 60 min gous Recombination by Acting on Defined Intermediates (A) Homologous and homeologous strand ex- change using bacteriophage DNA. The numbering system of the DNA species, schematically de- 3 picted to the right of the gel, reflects their order of 5 appearance. See also Table S1. 0 nm 4 Branched Intermediate 4 (B) Defined reaction intermediates consist of one 3.0 nm ssDNA and one dsDNA molecule. Gel-purified 6 intermediates 4 and 5 from the homeologous 1 reaction were incubated with SSB to mark ssDNA regions and were analyzed with SFM. Images are 0.6 3 0.6 mm, with z dimension indicated by the colored bar. Regions of dsDNA appear as a thin 7 blue line; SSB-coated ssDNA appears as a thick 2 yellow line. Drawings next to the SFM images are 0 nm interpretations of the intermediates, with one cir- cular fd ssDNA (red) and one linear M13 dsDNA CD (black). ssDNA gap length Homeologous (C) Intermediate 4 has a shorter heteroduplex - +MutS-MutLregion than intermediate 5. Upper panel, histo- 0 45 90 135 180 0 45 90 135 180 min gram showing contour length measurements of the SSB-coated ssDNA gap (bold line in cartoon) in intermediates 4 (n = 48, red bars) and 5 (n = 116, blue bars); lower panel, histogram showing the contour length measurement of the linear dsDNA tail (bold line in cartoon) from intermediates 4 5 (n = 28, red bars) and 5 (n = 96, blue bars). (D) The effect of MutS (75 nM) and MutL (75 nM) 4 on homeologous strand exchange. See also 6 Figure S1. dsDNA tail length 1 7 2 RESULTS Characterization of Recombination Intermediates Formed during RecA-Mediated Homeologous Strand Exchange To mechanistically study MMR-directed inhibition of homeologous recombination, dissolves the recombination intermediate. Interestingly, in bio- we established and characterized RecA-mediated strand-ex- chemical experiments UvrD can both stimulate and prevent change reactions using the 6.4 kb genomes of bacteriophage RecA-mediated homologous strand exchange (Morel et al., fd and M13 as DNA substrates. In the homologous reaction, 1993). Furthermore, UvrD dismantles RecA nucleoprotein fila- linear dsDNA (designated as species 1 in Figure 1A) and circular ments, thereby preventing joint molecule formation (Veaute ssDNA (species 2) substrates from the bacteriophage M13 were et al., 2005). How these activities are regulated within the con- used. The homeologous reaction was performed between fd cir- text of homeologous recombination remains to be determined. cular ssDNA and M13 linear dsDNA, whose DNA sequences The second mechanism is MutH dependent and explains diverge by approximately 3% (Table S1 available online). Both events occurring during the late stage of homeologous re- homologous and homeologous reactions form DNA intermedi- combination requiring MutS, MutL, MutH, DNA helicase, and ates (species 3, 4, and 5) before products of nicked circular de novo DNA synthesis. Both mechanisms have yet to be tested dsDNA (species 6 in Figure 1A) and linear ssDNA (species 7) biochemically. are generated. Here, we address how MutS and MutL inhibit strand exchange Based on structural analysis of DNA intermediates formed by trapping homeologous recombination intermediates and how during strand exchange with RecA and ATPgS(Menetski et al., UvrD helicase is directed to resolve the trapped intermediates. 1990) and Rad51 (Holmes et al., 2002), intermediates 3, 4, and Our results provide new insights into the dynamic interplay 5 observed in our system are expected to be joint molecules. between these two DNA repair systems with implications for Knowledge of the exact structure and stoichiometry of these eukaryotic antirecombination. intermediates is essential for a mechanistic explanation of the Molecular Cell 51, 326–337, August 8, 2013 ª2013 Elsevier Inc. 327 Molecular Cell Antirecombination Mechanism of DNA Mismatch Repair effect of MMR proteins on strand exchange; thus, we examined addressing strand invasion, homologous pairing, and strand the structures of DNA intermediates 4 and 5 from the homeolo- exchange in the absence of extensive branch migration. We gous reaction (red and blue boxes, Figure 1A) with scanning used 50-labeled linear ssDNA (90 nt), of which 54 nucleotides force microscopy (SFM). We incubated purified DNA intermedi-
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