PMID:17823128

Citation	Webb, MR, Plank, JL, Long, DT, Hsieh, TS and Kreuzer, KN (2007) The phage T4 protein UvsW drives Holliday junction branch migration. J. Biol. Chem. 282:34401-11
Abstract	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.
Links	PubMed PMC2094049 Online version:10.1074/jbc.M705913200
Keywords	Amino Acid Substitution; Bacteriophage T4/physiology; DNA Damage/physiology; DNA Helicases/chemistry; DNA Helicases/genetics; DNA Helicases/metabolism; DNA Repair/physiology; DNA Replication/physiology; DNA, Cruciform/chemistry; DNA, Cruciform/genetics; DNA, Cruciform/metabolism; DNA, Single-Stranded/chemistry; DNA, Single-Stranded/genetics; DNA, Single-Stranded/metabolism; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/metabolism; Mutation, Missense; Recombination, Genetic/physiology; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism; Virus Replication/physiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPT4:UVSW	NOT	GO:0009379: Holliday junction helicase complex	ECO:0000315:		C		Figure 5: In the presence of ATP and wild type UvsW branch migration of DHJS-2 Holliday junction occurs, whereas branch migration does not occur in the presence of K141R mutant of UvsW and even in the presence of UvsW when ATP is absent.	complete CACAO 12663

References

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