Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme.
We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.
Dillingham
Mark S
MS
Section of Microbiology, Center for Genetics and Development, University of California, Davis, California 95616, USA.
Webb
Martin R
MR
Kowalczykowski
Stephen C
SC
D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D021122:Protein Subunits; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases