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.Homologous recombination plays essential, but seemingly paradoxical, roles in promoting genetic diversity through meiotic recombination and in maintaining genomic stability. The importance of recombination in genomic stability is ensured by its roles in the repair of DNA damage such as double strand breaks and in the restart of stalled replication forks (for review, see Ref. (1)). For all of these processes, the Holliday junction (HJ) 2 is a common feature. HJs can be formed through an enzyme-mediated process that involves the invasion of a singlestranded portion of a DNA into a homologous sequence in another DNA. HJs may also be formed through regression of an inactive replication fork. The branch point of a HJ can migrate through strand exchange, as long as both participating duplex segments are homologous. Branch migration (BM) has been shown to be catalyzed by a number of enzymes including RecG (2) and RuvAB (3) from Escherichia coli, a subset of eukaryotic RecQ enzymes (4, 5), and Rad54 from human cells (6).Bacteriophage T4 has been used as a model biological system since the early days of molecular biology and has been invaluable in advancing our understanding of many fundamental biological processes. The T4 genome encodes ϳ300 different proteins, including all essential enzymes at the replication fork, making it a relatively simple organism to study (7). Furthermore, T4 proteins show about as much sequence homology to eukaryotes as they do to prokaryotes (8). For example, T4 DNA polymerase (gp43)...