title: Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding.
abstract: 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616-8665; Department of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665; Biophysics Graduate Group, University of California, Davis, CA 95616-8665.
authors.forename: Behzad
authors.initials: B
authors.lastname: Rad
authors.affiliation: Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616-8665; Department of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665;
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Forget
authors.affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665;
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Baskin
authors.affiliation: Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616-8665; Department of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665; Biophysics Graduate Group, University of California, Davis, CA 95616-8665 sckowalczykowski@ucdavis.edu.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Kowalczykowski
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D008853:Microscopy / Q000379:methods; D009690:Nucleic Acid Conformation; D053484:RecQ Helicases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D005456:Fluorescent Dyes; D053484:RecQ Helicases
keywords: BLM; DNA repair; TIRF microscopy; fluorescence; recombination