Mechanism of translocation and kinetics of DNA unwinding by the helicase RecG.
RecG is a DNA helicase involved in the repair of damage at a replication fork and catalyzes the reversal of the fork to a point beyond the damage in the template strand. It unwinds duplex DNA in reactions that are coupled to ATP hydrolysis. The kinetic mechanism of duplex DNA unwinding by RecG was analyzed using a quantitative fluorescence assay based on the process of contact quenching between Cy3 and Dabcyl groups attached to synthetic three-way DNA junctions. The data show that the protein moves at a rate of 26 bp s(-1) along the duplex DNA during the unwinding process. RecG ATPase activity during translocation indicates a constant rate of 7.6 s(-1), measured using a fluorescent phosphate sensor, MDCC-PBP. These two rates imply a movement of approximately 3 bp per ATP hydrolyzed. We demonstrate in several trapping experiments that RecG remains attached to DNA after translocation to the end of the arm of the synthetic DNA junction. ATPase activity continues after translocation is complete. Dissociation of RecG from the product DNA occurs only very slowly, suggesting strong interactions between them. The data support the idea that interactions of the duplex template arm with the protein are the major sites of binding and production of translocation.
Martinez-Senac
Maria M
MM
MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Webb
Martin R
MR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry; D003374:Coumarins / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D028044:Phosphate-Binding Proteins / Q000737:chemistry; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D020124:Thermotoga maritima / Q000201:enzymology*; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000737:chemistry
D002232:Carbocyanines; D003374:Coumarins; D009841:Oligonucleotides; D028044:Phosphate-Binding Proteins; D010710:Phosphates; C089187:cyanine dye 3; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D000255:Adenosine Triphosphate; D004247:DNA; D004124:p-Dimethylaminoazobenzene; D000251:Adenosine Triphosphatases; D004265:DNA Helicases