Kinetics of ATP-stimulated nuclease activity of the Escherichia coli RecBCD enzyme.
The RecBCD enzyme is an ATP-dependent nuclease on both single-stranded and double-stranded DNA substrates. We have investigated the kinetics of the RecBCD-catalyzed reaction with small, single-stranded oligodeoxyribonucleotide substrates under single-turnover conditions using rapid-quench flow techniques. RecBCD-DNA complexes were allowed to form in pre-incubation mixtures. The nuclease reactions were initiated by mixing with ATP. The reaction time-courses were fit to several possible reaction mechanisms and quantitative estimates were obtained for rate constants for individual reaction steps. The relative rates of forward reaction versus dissociation from the DNA, and the fact that inclusion of excess non-radiolabeled single-stranded DNA to trap free RecBCD has no effect on the nuclease reaction, indicates that the reaction is processive. The reaction products show that the reaction begins near the 3'-end of the [5'-32P]DNA substrates and the major cleavage sites are two to four phosphodiester bonds apart. The product distribution is unchanged as the ATP concentration varies from 10 microM to 100 microM ATP, while the overall reaction rate varies by about tenfold. These observations suggest that DNA cleavage is tightly coordinated with movement of the enzyme along the DNA. The reaction time-courses at low concentrations of ATP (10 microM and 25 microM) have a significant lag before cleavage products appear. We propose that the lag represents ATP-dependent movement of the DNA from an initial binding site in the helicase domain of the RecB subunit to the nuclease active site in a separate domain of RecB. The extent of reaction of the substrate is limited (approximately 50%) under all conditions. This may indicate the formation of a non-productive RecBCD-DNA complex that does not dissociate in the 1-2 s time-scale of our experiments.
Ghatak
Archana
A
Department of Chemistry & Biochemistry, University of Maryland, College Park, MD 20742, USA.
Julin
Douglas A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D013997:Time Factors
D009841:Oligonucleotides; D000255:Adenosine Triphosphate; D004247:DNA; D043211:Exodeoxyribonuclease V