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

Abstract

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) andori(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 atori(uvsY) persist longer in a uvsWmutant 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.

ACKNOWLEDGMENTS

We thank Leslie Derr, Kelly Carles-Kinch, Helen Kreuzer, and Karyn Belanger for providing the foundation for this study.

This work was supported by grant GM34622 from the National Institutes of Health to K.N.K. and by NIH fellowship F32 GM19000 to K.C.D.