title: Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD-DNA complexes.
abstract: RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.
authors.affiliation: Department of Physics, Amherst College, Amherst, MA 01002, USA.
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Carter
authors.affiliation: Department of Physics, University of Colorado, Boulder, CO 80309, USA JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309, USA.
authors.forename: Maasa H
authors.initials: MH
authors.lastname: Seaberg
authors.affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 11221, Taiwan Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H4B1R6, Canada.
authors.forename: Hsiu-Fang
authors.initials: HF
authors.lastname: Fan
authors.affiliation: Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Gang
authors.initials: G
authors.lastname: Sun
authors.affiliation: Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Wilds
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H4B1R6, Canada.
authors.forename: Hung-Wen
authors.initials: HW
authors.lastname: Li
authors.affiliation: JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309, USA Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA tperkins@jila.colorado.edu.
authors.forename: Thomas T
authors.initials: TT
authors.lastname: Perkins
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C087189:2-fluoro-ADP; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli