From the Cover: PNAS Plus: Single-molecule visualization of RecQ helicase reveals DNA melting,nucleation, and assembly are required for processive DNA unwinding

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.DNA helicases are ubiquitous enzymes involved in many aspects of DNA metabolism, including DNA replication, repair, and recombination. These enzymes work by coupling the hydrolysis of nucleoside triphosphates (NTPs) to unwinding of double-stranded DNA (dsDNA) to produce single-stranded DNA (ssDNA) (1). This activity allows the cell’s machinery to access the information stored within the bases of the double helix. RecQ helicase from Escherichia coli is the founding member of the RecQ family of helicases (2). These enzymes belong to the superfamily 2 (SF2) group of helicases, yet share greater sequence homology with their own family members, and they play important roles in the maintenance of genomic integrity by DNA recombination and repair (1, 3). Mutations in the human RecQ-like helicases, Bloom (BLM), Werner (WRN), and RecQ4 proteins, lead to Bloom’s, Werner’s, and Rothmund–Thomson syndromes, respectively. These genetic disorders are characterized by genomic instability and an increased incidence of cancers (4).E. coli RecQ is a 3′ → 5′ helicase that functions in DNA-break repair by homologous recombination (2, 5, 6). RecQ and RecJ, a 5′ → 3′ exonuclease, process ssDNA gaps or dsDNA breaks into ssDNA for recombinational repair by RecA (7–10). In addition, RecQ ensures recombination fidelity in vivo by removing inappropriately paired joint molecules to prevent illegitimate recombination and also by disrupting joint molecule intermediates to facilitate repair by synthesis-dependent strand annealing, preventing chromosomal crossing over (7, 9, 11, 12). RecQ also functions with topoisomerase III (Topo III), a type I topoisomerase, to catenate and decatenate DNA molecules and to separate converged replication forks (13, 14). RecQ and Topo III provide an alternative to the RuvABC pathway for disengaging double Holliday junctions and do so without producing chromosomal crossovers (15).In vitro, RecQ can unwind a multitude of DNA substrates and does not require a ssDNA tail, or even a dsDNA end, to initiate unwinding; consequently, it is distinctive in being able to unwind covalently closed circular plasmid DNA (16, 17). The winged-helix domain of RecQ is important for the recognition of this broad array of DNA substrates (18). This domain binds to dsDNA yet it adopts a flexible conformation that allows it to adapt to many DNA structures. A curious feature of RecQ is that maximal unwinding requires nearly stoichiometric amounts of protein relative to the DNA (one protein per ∼10 bp), which can be partially mitigated (one protein per ∼30 bp) by including the ssDNA binding protein, SSB (5, 6, 16, 19). This behavior is compatible with the low processivity of ssDNA translocation (∼30–100 nucleotides) by RecQ (20–22) and other RecQ members (23). Paradoxically, at limiting concentrations, most RecQ helicases nonetheless efficiently unwind several kilobases of dsDNA in the course of their normal functions (9, 16, 24–26), suggesting a dynamic unwinding process. Although RecQ can unwind DNA as a monomer, it also shows a functional cooperativity when unwinding DNA with an ssDNA tail (27–29). Thus, multiple monomers can bind to ssDNA to unwind long dsDNA regions.Fluorescence techniques coupled with single-molecule microscopy have emerged as a powerful method for studying the unwinding mechanism of helicases (30–35). These assays measure individual enzymes directly or indirectly through their actions on individual DNA molecules, thus alleviating many of the drawbacks of ensemble experiments. In particular, total internal reflection fluorescence (TIRF) microscopy permits the detection of individual fluorophores with high sensitivity in the presence of a high background (30). To visualize the activity of DNA binding proteins and helicases, TIRF microscopy can be coupled with microfluidic techniques to facilitate visualization of molecules and exchange of solution components (36–39).Ensemble assays have elucidated many features of DNA unwinding by the RecQ helicase family, but the need to average over a heterogeneous and unsynchronized population of enzymes has precluded a thorough understanding of this diverse and universally important helicase family. To permit a more insightful analysis, we directly visualized unwinding of individual molecules of DNA by RecQ helicase. Unwinding was monitored using fluorescent SSB to visualize generation of ssDNA. On single DNA molecules, we could see tracks of the fluorescent SSB binding to ssDNA produced by the helicase activity of RecQ. We show that RecQ initiates DNA unwinding via melting of duplex DNA at internal sites. Once initiated, DNA unwinding propagates either uni- or bidirectionally via the cooperative action of multiple RecQ molecules at the junction of ssDNA with dsDNA. Collectively, these observations define a stable oligomeric complex of subunits involved in processive helicase action, which is concordant with both biochemical and biological function of RecQ helicases and other helicases.