Crystallographic and NMR Analyses of UvsW and UvsW.1 from Bacteriophage T4

Luke A. Knox,Iain D. Kerr,Sivashankar Sivakolundu,Stephen W. White,Jeffrey C. Buchsbaum,Zhenmei Li,Richard Kriwacki

doi:10.1074/jbc.m705900200

Abstract

The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3'-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7A and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases.

Highlights

Bacteriophage T4 remains an ideal model system in which to study the fundamental mechanisms of nucleic acid metabolism
It has been known for some time that stalled and collapsed replication forks can lead to DNA double-strand breaks [6], and recombination-dependent replication (RDR) has a pivotal role in repairing the lesions generated by these potentially lethal events [7]
UvsW was first identified as a T4 genetic locus that controls genome stability, and mutations within the UvsW gene rendered the virus susceptible to DNA damage due to UV radiation and treatment with hydroxyurea [8, 10]. These observations suggested that UvsW has prominent roles in DNA double-strand break repair (DSBR), and the rescue of collapsed replication forks, respectively

Summary

Introduction

Bacteriophage T4 remains an ideal model system in which to study the fundamental mechanisms of nucleic acid metabolism. During the early stage of the T4 infection cycle, DNA synthesis is initiated by a classic origin-dependent mechanism in which replication complexes are assembled onto persistent R-loops (RNA-DNA hybrids) [2]. Homologous recombination is a key process in DNA metabolism that remains poorly understood at the molecular level, and T4 is an ideal system in which to study the mechanism due to its central role in RDR and T4 replication. UvsX and UvsY are both members of the uvsWXY system, which is associated with susceptibility to DNA damage because of UV radiation ( the name) This is consistent with the roles that recombination and RDR are known to play in the repair of DNA double-strand breaks [5]. UvsW is involved in the switch from early origin-dependent to late replication-dependent replication during the T4 infection cycle, and catalyzes the disassembly of R-loops that act as primers at replication origins [16]

Methods

Results

Conclusion