RecQ helicase and RecJ nuclease provide complementary functions to resect DNA for homologous recombination

Recombinational DNA repair by the RecF pathway of Escherichia coli requires the coordinated activities of RecA, RecFOR, RecQ, RecJ, and single-strand DNA binding (SSB) proteins. These proteins facilitate formation of homologously paired joint molecules between linear double-stranded (dsDNA) and supercoiled DNA. Repair starts with resection of the broken dsDNA by RecQ, a 3′→5′ helicase, RecJ, a 5′→3′ exonuclease, and SSB protein. The ends of a dsDNA break can be blunt-ended, or they may possess either 5′- or 3′-single-stranded DNA (ssDNA) overhangs of undefined length. Here we show that RecJ nuclease alone can initiate nucleolytic resection of DNA with 5′-ssDNA overhangs, and that RecQ helicase can initiate resection of DNA with blunt-ends or 3′-ssDNA overhangs by DNA unwinding. We establish that in addition to its well-known ssDNA exonuclease activity, RecJ can display dsDNA exonuclease activity, degrading 100–200 nucleotides of the strand terminating with a 5′-ssDNA overhang. The dsDNA product, with a 3′-ssDNA overhang, is an optimal substrate for RecQ, which unwinds this intermediate to reveal the complementary DNA strand with a 5′-end that is degraded iteratively by RecJ. On the other hand, RecJ cannot resect duplex DNA that is either blunt-ended or terminated with 3′-ssDNA; however, such DNA is unwound by RecQ to create ssDNA for RecJ exonuclease. RecJ requires interaction with SSB for exonucleolytic degradation of ssDNA but not dsDNA. Thus, complementary action by RecJ and RecQ permits initiation of recombinational repair from all dsDNA ends: 5′-overhangs, blunt, or 3′-overhangs. Such helicase–nuclease coordination is a common mechanism underlying resection in all organisms.Homologous recombination is a relatively error-free mechanism to repair double-stranded DNA (dsDNA) breaks (DSBs) and single-stranded DNA (ssDNA) gaps, which are produced by UV light, γ-irradiation, and chemical mutagens (1). In wild-type Escherichia coli, the labor of recombinational repair is divided between the RecBCD and RecF pathways of recombination, which are responsible for the repair of DSBs and ssDNA gaps, respectively (2–5). However, the proteins of the RecF pathway are capable of DSB repair, as well as ssDNA gap repair: in recBC mutant cells containing the suppressor mutations, sbcB and sbcC (suppressors of recBC), the proteins of the RecF pathways provide the needed recombinational DNA repair functions (2, 6).The RecF pathway in E. coli involves the functions of RecA, RecF, RecG, RecJ, RecN, RecO, RecQ, RecR, RuvA, RuvB, RuvC, and single-strand DNA binding (SSB) proteins (1, 7). The RecF pathway of recombination is evolutionarily conserved across Bacteria, with most of components present in all bacteria (8). In addition, orthologs of RecF pathway proteins are found in Eukarya. RecA promotes DNA strand invasion and exchange (9–11), as does eukaryotic Rad51 (12, 13). RecO can both anneal SSB–ssDNA complexes (14, 15) and, in conjunction with RecR (and RecF), mediate loading of RecA onto SSB–ssDNA complexes (16–18). Saccharomyces cerevisiae Rad52 is a functional homolog of RecO in that it also displays both DNA-annealing and Rad51-loading activities (19–22). The RecFOR complex promotes the loading of RecA onto SSB-coated gapped DNA at ssDNA–dsDNA junctions (17, 18) and, when mutated, is suppressed by hyperactive alleles of recA (23), a property that is shared with the yeast Rad55/57 proteins (24). Furthermore, human BRCA2 protein and a fungal analog, Brh2, are partial functional analogs of the RecFOR proteins (25–27).RecQ helicase plays several roles in both early and late steps of recombination (28, 29), as do the RecQ-family helicases in Eukarya [e.g., Sgs1 and Bloom Syndrome helicase (BLM)] (30–32). In addition, eukaryotic Exonuclease 1 (Exo1) and Dna2 helicase/nuclease function somewhat analogously, although not identically, to RecJ nuclease (33–36). The in vitro reconstitution of DSB repair in E. coli, yeast, and human have shown that resection involves specific pairs of a helicase and nuclease for DNA end resection: RecQ/RecJ, Sgs1/Dna2, BLM/DNA2, and BLM/EXO1 (28, 37–39).A comparison of DSB repair by the RecBCD and RecF pathways shows that repair starts with the processing a DSB into resected dsDNA with a 3′-ssDNA overhang (7). RecJ has a 5′ to 3′ exonuclease activity on ssDNA and the action of RecJ is facilitated by RecQ, which has a 3′ to 5′ helicase activity (40, 41). The resulting processed DNA has a 3′-ssDNA overhang. The RecFOR complex binds to the 5′-end at the junction between ssDNA and dsDNA, and loads RecA protein onto the adjacent ssDNA (17, 18). Finally, the RecA nucleoprotein filament promotes pairing with homologous dsDNA (9). These steps have been reconstituted in vitro in a coordinated reaction using RecAFORQJ and SSB proteins (28).Despite progress, most studies have used DNA substrates with simple blunt-ends. However, in vivo, there are many potential structures at the end of a DSB. When the DSB is created by a replication fork encountering nicked DNA, the break can be blunt-ended (5). However, related mechanisms can produce dsDNA with either 5′- or 3′-ssDNA overhangs. Similarly, the actual intermediates of DNA processing may result in dsDNA with either 5′- or 3′-ssDNA ends. Clearly, a DNA repair pathway must be capable of dealing with such a variety of DNA end structures. In this study, we investigated the processing of DSBs by RecJ and RecQ, both individually and together. We found that a DNA with a 5′-ssDNA overhang end was degraded by RecJ nuclease and converted into an intermediate with a 3′-ssDNA overhang. Although this intermediate was no longer a substrate for RecJ, RecQ could bind to this intermediate and initiate unwinding, thereby supplying 5′-tailed ssDNA for further resection by RecJ. In addition, we established that RecQ allows RecJ to initiate nucleolytic resection on otherwise poor substrates (e.g., blunt-end DNA or DNA with 3′-ssDNA overhangs). Thus, RecQ and RecJ cooperate biochemically to create DNA intermediates for one another that enable resection of all types of broken DNA molecules.