Kinetics of DNA unwinding by the RecD2 helicase from Deinococcus radiodurans.
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 k(unw) = 5.5 s(-1) and a dissociation step from partially unwound DNA of k(off) = 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.
Shadrick
William R
WR
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Julin
Douglas A
DA
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002845:Chromatography / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D034301:Deinococcus / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D043211:Exodeoxyribonuclease V / Q000737:chemistry* / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D009841:Oligonucleotides / Q000737:chemistry; D011489:Protein Denaturation
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D004247:DNA; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases; D005976:Glutaral