Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

• Published in: The Journal of biological chemistry 2010-06, Vol.285 (23), p.17292-17300
• Main Authors: Shadrick, William R., Julin, Douglas A.
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - metabolism; Chromatography - methods; Cross-Linking Reagents - chemistry; Deinococcus - metabolism; Deinococcus radiodurans; Dimerization; DNA - chemistry; DNA and Chromosomes; DNA Enzymes; DNA Helicase; DNA Helicases - chemistry; DNA Helicases - metabolism; DNA Recombination; DNA Repair; DNA, Single-Stranded - chemistry; Dose-Response Relationship, Drug; Enzyme Kinetics; Enzymology; Escherichia coli; Exodeoxyribonuclease V - chemistry; Exodeoxyribonuclease V - metabolism; Glutaral - chemistry; Kinetics; Models, Biological; Oligonucleotides - chemistry; Protein Denaturation; RecBCD Enzyme
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.111427

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of kunw = 5.5 s−1 and a dissociation step from partially unwound DNA of koff = 1.9 s−1. These results indicate a kinetic step size of about 3–4 bp, unwinding rate of about 15–20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5′-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.