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End Resection at Double-Strand Breaks: Mechanism and Regulation

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End Resection at Double-Strand Breaks: Mechanism and Regulation Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press End Resection at Double-Strand Breaks: Mechanism and Regulation Lorraine S. Symington Department of Microbiology and Immunology,Columbia University Medical Center, New York, New York 10032 Correspondence: [email protected] RecA/Rad51 catalyzed pairing of homologous DNA strands, initiated by polymerization of the recombinase on single-stranded DNA (ssDNA), is a universal feature of homologous recombination (HR). Generation of ssDNA from a double-strand break (DSB) requires nu- cleolytic degradation of the 50-terminated strands to generate 30-ssDNA tails, a process referred to as 50 –30 end resection. The RecBCD helicase–nuclease complex is the main end-processing machine in Gram-negative bacteria. Mre11-Rad50 and Mre11-Rad50- Xrs2/Nbs1 can play a direct role in end resection in archaea and eukaryota, respectively, by removing end-blocking lesions and act indirectly by recruiting the helicases and nucle- ases responsible for extensive resection. In eukaryotic cells, the initiation of end resection has emerged as a critical regulatory step to differentiate between homology-dependent and end- joining repair of DSBs. SBs can arise accidentally during normal must first be degraded to generate long 30- Dcell metabolism or after exposure of cells ssDNA tails, a process referred to as 50 –30 end to DNA-damaging agents, and also serve as in- resection. The 30-ssDNA tails are then bound by termediates in a numberof programmed recom- a member of the RecA/Rad51 family of proteins bination events in eukaryotic cells (Mehta and to initiate homologous pairing and serve as Haber 2014). The repair of DSBs is critical for primers for DNA synthesis following strand in- maintenance of genome integrity, and misre- vasion. Strand invasion intermediates are fur- pair, or failure to repair, is associated with chro- ther processed by helicases and/or nucleases mosome rearrangements, chromosome loss, or (Bizard and Hickson 2014; Wyatt and West even cell death. Both prokaryotic and eukaryotic 2014), and ultimately by gap-filling DNA syn- cells have evolved elaborate mechanisms for the thesis and ligation, to generate mature recombi- recognition and repair of DSBs. The two pre- nant products. The DNA end-resection step of dominant repair mechanisms are HR and non- HR is conserved in all domains of life, but the homologous end joining (NHEJ). HR relies on mechanisms used for generating ssDNA are dis- the presence of an intact homologous duplex to tinct. Here, we review the basic machinery for template repair of the broken strands, whereas DNA end resection in bacteria, archaea, and eu- NHEJ repairs DSBs by direct ligation of the DNA karyota and the regulation of end resection in ends. For DSBs to be repaired by HR, the ends eukaryotic cells. Editors: Stephen Kowalczykowski, Neil Hunter, and Wolf-Dietrich Heyer Additional Perspectives on DNA Recombination available at www.cshperspectives.org Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016436 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016436 1 Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press L.S. Symington END RESECTION IN BACTERIA (Taylor and Smith 2003). RecD is the fast, or lead, motor on the 50-terminated strand, where- The heterotrimeric RecBCD nuclease is the ma- as RecB translocates more slowly on the 30-ter- jor end-processing machine in Escherichia coli minated strand until the complex encounters a and is conserved across the majority of Gram- Chi site (Fig. 1). Upon Chi recognition, the en- negative bacteria (Dillingham and Kowalczy- zyme pauses, the RecD subunit is inactivated, kowski 2008). RecBCD is a complex enzyme and continued unwinding is driven by the that couples ATP-dependent unwinding to RecB helicase, resulting in a slower translocation DNA degradation (Smith 2001; Dillingham and rate (Spies et al. 2003). Before encountering Chi, Kowalczykowski 2008). The potent exonuclease the 30 end is more extensivelycleaved by the RecB activity of RecBCD can degrade thousands of endonuclease than the 5-terminated strand, but bases per second. This destructive activity of after Chi recognition, degradation of the 30 end RecBCD plays an important role in protecting is suppressed, and cleavage of the 50-terminated bacteria from invading bacteriophages with lin- strand is stimulated, generating a 30-ssDNA tail ear genomes. Nuclease activity resides in the (Anderson and Kowalczykowski 1997a). In ad- carboxy-terminal region of the RecB subunit dition, RecB facilitates loading of RecA onto the and is regulated by RecC and by interaction 30-terminated strand after Chi recognition (An- with a specific sequence called Chi (50- derson and Kowalczykowski 1997b). How does GCTGGTGG-30) (Wang et al. 2000). Chi sites ChiregulatethenucleaseactivitiesoftheRecBCD suppress the nuclease activity of RecBCD and complex? Structural studies indicate that a “pin” stimulate recombination locally (Lam et al. in RecC separates the strands of duplex DNA en- 1974; Dixon and Kowalczykowski 1993). The tering the complex driven by the RecB and RecD 8-bp nonpalindromic Chi sites are overrepre- translocases (Singleton et al. 2004). As the sepa- sented in the E. coli genome and are oriented rated strands passthrough the RecBCD complex, toward the replication origin such that loading the RecC subunit recognizes Chi, resulting in a of RecBCD at a collapsed replication fork would conformational change that opens a molecular lead to suppression of DNA degradation upon latch allowing the 30-terminated strand to bypass Chi recognition by RecBCD and activation of the RecB nuclease domain and exit the complex HR (Blattner et al. 1997). (Handa et al. 2012; Yanget al. 2012). Our current view of how RecBCD promotes The RecBC enzyme behaves similarly to recombination derives from a combination of Chi-modified RecBCD. RecBC unwinds dou- bulk-phase biochemistry, single-DNA molecule ble-stranded DNA (dsDNA) more slowly than imaging, electron microscopy (EM), and struc- RecBCD and constitutively loads RecA onto the tural studies. RecBCD binds with high affinity to 30 end of the unwound strands. Consistent with blunt or nearly blunt-ended linear duplex DNA the in vitro studies, recD mutants are recombi- (Taylor and Smith 1985). Unwinding is driven nation proficient and recombination is stimu- by the RecB and RecD subunits, which are hel- lated at ends instead of in the vicinity of Chi sites icases with opposite polarities and thus translo- (Thaler et al. 1989; Churchill et al. 1999). By cate the complexon both strands of duplex DNA contrast, recB and recC mutants show high in the same direction (Dillingham et al. 2003; sensitivity to X rays and low frequency of recom- Taylor and Smith 2003). The robust translocase bination as measured by conjugation or trans- activity of the RecBCD complex is able to dis- duction (Persky and Lovett 2008). However, place tightly bound proteins from duplex DNA these defects can be suppressed by inactivation (Finkelstein et al. 2010). Under conditions in of the 30 exonucleases ExoI and SbcCD, suggest- which the nuclease activity of the complex is ing that an alternative mechanism is able to minimized, the enzyme unwinds duplex DNA generate 30-ssDNA tailed intermediates in the to produce one long 50-ssDNA tail and an ssDNA absence of RecBCD, but the ends are unstable loop associated with a short 30-ssDNAtail owing because of 30 nuclease activity. Recombination to the two helicases operating at different speeds in the recBC-suppressed strains is caused by the 2 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016436 Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Processing of DNA Breaks: Mechanism and Regulation RecBCD Chi Slow motor RecB 3′–5′ helicase 5′ RecB nuclease 3′ Fast motor RecD 5′–3′ helicase RecC 3′ 5′ Pause at Chi 3′ 5′ RecA loading Slow motor 3′ 5′ Figure 1. End processing by the RecBCD complex. RecBCD loads at ends and translocates on both strands using the RecD and RecB helicase subunits. RecB degrades both DNA strands exiting the complex, but with more incisions on the 30 strand than the 50-terminated strand. RecBCD pauses at a Chi site, and the RecD subunit is modified; continued translocation is driven by the RecB helicase. After Chi recognition, RecB directs loading of RecA onto the 30 end and degrades only the 50 strand. RecF pathway of recombination, which normal- 2014). recJ and recQ mutants show UV sensitiv- ly functions during ssDNA gap repair (Persky ityandmay be required to expand ssDNAgapsto and Lovett 2008). Resection by the RecF path- facilitateRecAbinding(PerskyandLovett 2008). way requires the 50 –30 exonuclease, RecJ, and is RecJ can also cooperate with RecB and RecC in stimulated by the RecQ 30 –50 helicase and the the absence of RecD (Lovett et al. 1988; Dermic ssDNA-binding protein, SSB (Han et al. 2006; 2006). The high frequency of conjugal recombi- Handa et al. 2009). RecJ requires an ssDNA tail nation observed in recD mutants is reduced by of .6 nucleotides for binding and degrades to mutation of recJ, but not by recQ. The residual the ssDNA–dsDNA junction, releasing mono- recombination observed in the recD recJ mutant nucleotide products (Han et al. 2006). Although requires ExoVII, which degrades ssDNA from 50 originally characterized biochemically
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