Translocation of E. coli RecQ Helicase on Single-Stranded DNA

Helicases couple the hydrolysis of ATP to unidirectional movement on single-stranded DNA (ssDNA). Upon encountering double-stranded DNA (dsDNA), these ATP driven motors force the displacement of the opposite strand (1, 2), allowing essential metabolic processes such as DNA recombination, repair, and replication to proceed. Translocation on ssDNA also serves other biological roles such as displacing bound proteins to regulate pathways such as DNA recombination (3–5). The RecQ helicase from E. coli is a Super-Family 2 (SF2) helicase involved in the repair and recombination of DNA (6, 7). This protein is the founding member of the RecQ protein family of helicases, which is a family of highly conserved motor proteins in bacteria and eukaryotes. recQ was identified as a gene that conferred resistance to thymine-less growth and was found to function in recombinational DNA repair by the RecF pathway (8). RecQ, in conjunction with RecJ, processes DNA at the site of a stalled replisome to allow daughter-strand gap repair and recombination-mediated restart of DNA replication (9). RecQ, a 3′ to 5′ helicase (10), and RecJ, a 5′ to 3′ exonuclease (11), can process double-strand breaks to produce 3′-terminated ssDNA (12). This ssDNA serves as the substrate for assembly of a RecA filament, which then finds homology in intact dsDNA and promotes pairing of the ssDNA to the homologous target. RecQ has additional roles in DNA recombination and replication. RecQ can disrupt joint molecules both in vivo (13) and in vitro (12, 14). In addition, RecQ can function with Topoisomerase III (Topo III), a type I topoisomerase, to catenate and decatenate dsDNA (15, 16). This reaction can serve two potential functions: one is to dissolve double Holliday junctions formed during recombination (17–20), and the other is to decatenate converged replication forks (21). In vitro, the RecQ helicase binds to and unwinds a variety of different DNA substrates (10, 14, 22, 23). DNA with an ssDNA tail, gapped DNA, blunt dsDNA and covalently closed dsDNA are all unwound by RecQ, indicating that the helicase can function on a variety of intermediates found in DNA metabolism. RecQ is one of the few helicases that can unwind covalently closed, circular DNA—showing that it does not require a DNA end to enter dsDNA (15, 24). The ssDNA binding protein (SSB) from E. coli interacts with RecQ and stimulates its helicase activity to the extent that RecQ can unwind plasmid-length DNA (21, 24, 25). Initially, helicase assays performed with plasmid DNA suggested a nearly stoichiometric mechanism for unwinding dsDNA by RecQ (10, 22). In this mechanism, short patches of dsDNA are unwound by RecQ translocation over relatively short distances (24). However, unwinding of dsDNA is stimulated by SSB. In the presences of SSB, optimal unwinding requires 1 RecQ for every 30 base pairs, indicating that each helicase is capable of unwinding a region of DNA longer than its DNA binding site size (24, 26). Furthermore, experiments with dsDNA possessing a 3′-ssDNA tail show that RecQ alone can unwind dsDNA up to 25 base-pairs in length (27). The translocation mechanism of RecQ, however, has not been studied in as great detail as the unwinding mechanism. Several assays, as well as models, exist for the study of translocation by helicases on ssDNA. Steady-state analysis of several helicases on ssDNA has revealed estimates of processivity, directionality, and sequence effects on translocation (28–30). Kinetic mechanisms of translocation can be elucidated from analysis of the steady-state ATP hydrolysis activity. To understand translocation by RecQ, we studied the steady-state behavior of its ssDNA-dependent ATP hydrolysis activity as a function of DNA length. We find that the activity of RecQ increases with longer lengths of ssDNA. Moreover, when studied on ssDNA that is coated with T4 gene 32 protein (G32P) to limit the size of ssDNA available for activity, ATP hydrolysis was reduced in a manner that is quantitatively consistent with RecQ translocation on the ssDNA gaps that were available. We present a model to describe the mechanism of translocation for RecQ.