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Molecular and Cellular Biology, April 2001, p. 2706-2715, Vol. 21, No. 8
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.8.2706-2715.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

UvsW Protein Regulates Bacteriophage T4 Origin-Dependent Replication by Unwinding R-Loops

Kathleen C. Dudas and Kenneth N. Kreuzer*

Departments of Microbiology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received 31 October 2000/Returned for modification 30 November 2000/Accepted 31 January 2001

The UvsW protein of bacteriophage T4 is involved in many aspects of phage DNA metabolism, including repair, recombination, and recombination-dependent replication. UvsW has also been implicated in the repression of origin-dependent replication at late times of infection, when UvsW is normally synthesized. Two well-characterized T4 origins, ori(uvsY) and ori(34), are believed to initiate replication through an R-loop mechanism. Here we provide both in vivo and in vitro evidence that UvsW is an RNA-DNA helicase that catalyzes the dissociation of RNA from origin R-loops. Two-dimensional gel analyses show that the replicative intermediates formed at ori(uvsY) persist longer in a uvsW mutant infection than in a wild-type infection. In addition, the inappropriate early expression of UvsW protein results in the loss of these replicative intermediates. Using a synthetic origin R-loop, we also demonstrate that purified UvsW functions as a helicase that efficiently dissociates RNA from R-loops. These and previous results from a number of studies provide strong evidence that UvsW is a molecular switch that allows T4 replication to progress from a mode that initiates from R-loops at origins to a mode that initiates from D-loops formed by recombination proteins.

* Corresponding author. Mailing address: Department of Microbiology, Box 3020, Jones Building, Room 228, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-6466. Fax: (919) 681-8911. E-mail: kenneth.kreuzer{at}duke.edu.

Molecular and Cellular Biology, April 2001, p. 2706-2715, Vol. 21, No. 8
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.8.2706-2715.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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