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J. Biol. Chem., Vol. 282, Issue 47, 34401-34411, November 23, 2007

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The Phage T4 Protein UvsW Drives Holliday Junction Branch Migration^*

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.

Received for publication, July 19, 2007 , and in revised form, September 4, 2007.

^* This work was supported by National Institutes of Health Grants R01 GM066934, R01 GM29006, and 5T32CA009111-30. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Box 3711, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-6466; Fax: 919-684-6525; E-mail: kenneth.kreuzer{at}duke.edu.

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