Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme

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

Research output: Contribution to journalArticle

39 Citations (Scopus)

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 ∼30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is ∼50%. More significantly, the processivity of translocation is substantially reduced by ∼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.

Original languageEnglish (US)
Pages (from-to)37069-37077
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number44
DOIs
StatePublished - Nov 4 2005

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Exodeoxyribonuclease V
Single-Stranded DNA
DNA
Enzymes
DNA Helicases
Mutant Proteins
Mutagenesis
Coloring Agents
Adenosine Triphosphate
Assays
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme. / Dillingham, Mark S.; Webb, Martin R.; Kowalczykowski, Stephen C.

In: Journal of Biological Chemistry, Vol. 280, No. 44, 04.11.2005, p. 37069-37077.

Research output: Contribution to journalArticle

Dillingham, Mark S. ; Webb, Martin R. ; Kowalczykowski, Stephen C. / Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 44. pp. 37069-37077.
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