Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

Mark S. Dillingham,Martin R. Webb,Stephen C. Kowalczykowski

doi:10.1074/jbc.m505520200

Mark S. Dillingham, Martin R. Webb + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m505520200

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Abstract

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.

Highlights

Encounters a correctly oriented DNA sequence called Chi
Because RecBCD-catalyzed DNA unwinding can be assayed independently by following Single-stranded DNA-binding protein (SSB) binding to the ssDNA products of the reaction, it was shown that the presence of the dye does not inhibit the enzyme translocation and that the assay provides an accurate measure of both the rate and amplitude of DNA unwinding by RecBCD enzyme [14]
RecBCD enzyme was pre-bound to linearized pBR322 plasmid DNA (4361 bp) that was saturated with Hoechst 33258 dye

Summary

Introduction

Encounters a correctly oriented DNA sequence called Chi (crossover hotspot instigator 5Ј-GCTGGTGG). The use of two DNA motors is potentially capable of generating more force than a single motor This feature might result in an increased forward translocation rate, under conditions in vivo, in which the translocating enzyme may encounter a variety of “roadblocks” (protein-nucleic acid complexes) on its path to a properly aligned Chi sequence; RecBCD enzyme is sufficiently potent so it can displace nucleosomes from DNA [28]. To test these ideas directly, we studied the DNA translocation and unwinding activities of RecBCD enzymes in which either the RecB or RecD motor is inactivated by mutagenesis. This observation strongly suggests that the activities of the two motors are at least partially independent

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Conclusion