title: A nucleotide-dependent and HRDC domain-dependent structural transition in DNA-bound RecQ helicase.
abstract: The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF4. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.
authors.affiliation: From the Department of Biochemistry, ELTE-MTA "Momentum" Motor Enzymology Research Group, Eötvös University, Pázmány P. s. 1/c, H-1117 Budapest, Hungary.
authors.forename: Zsuzsa S
authors.initials: ZS
authors.lastname: Kocsis
authors.forename: Kata
authors.initials: K
authors.lastname: Sarlós
authors.forename: Gábor M
authors.initials: GM
authors.lastname: Harami
authors.forename: Máté
authors.initials: M
authors.lastname: Martina
authors.forename: Mihály
authors.initials: M
authors.lastname: Kovács
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009942:Organometallic Compounds / Q000737:chemistry; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D009942:Organometallic Compounds; C105164:adenosine diphosphate tetrafluoroaluminate; D000244:Adenosine Diphosphate; D043244:Ribonuclease III; C506724:RecQ protein, E coli; D053484:RecQ Helicases
keywords: ATPases; DNA Repair; DNA-Protein Interaction; Molecular Motors; Protein Conformation; Recombination; Structure