Kelly Carles-Kinch

Bacteriophage T4 UvsW protein is a helicase involved in recombination, repair and the regulation of DNA replication origins

Bacteriophage T4 UvsW protein is involved in phage recombination, repair and the regulation of replication origins. Here, we provide evidence that UvsW functions as a helicase. First, expression of UvsW allows growth of an (otherwise inviable) Escherichia coli recG rnhA double mutant, consistent with UvsW being a functional analog of the RecG helicase. Second, UvsW contains helicase sequence motifs, and a substitution (K141R) in the Walker ‘A’ motif prevents growth of the E.coli recG rnhA double mutant. Third, UvsW, but not UvsW‐K141R, inhibits replication from a T4 origin at which persistent RNA–DNA hybrids form and presumably trigger replication initiation. Fourth, mutations that inactivate UvsW and endonuclease VII (which cleaves DNA branches) synergistically block repair of double‐strand breaks. These in vivo results are consistent with a model in which UvsW is a DNA helicase that catalyzes branch migration and dissociation of RNA–DNA hybrids. In support of this model, a partially purified GST/UvsW fusion protein, but not a GST/UvsW‐K141R fusion, displays ssDNA‐dependent ATPase activity and is able to unwind a branched DNA substrate.

Role of recombinational repair in sensitivity to an antitumour agent that inhibits bacteriophage T4 type II DNA topoisomerase

The bacteriophage T4‐encoded type II DNA topoisomerase is the major target for the antitumour agent m‐AMSA (4‐(9‐acridinylamino)methanesulphon‐m‐anisidide) in phage‐infected bacterial cells. Inhibition of the purified enzyme by m‐AMSA results in formation of a cleavage complex that contains the enzyme covalently attached to DNA on both sides of a double‐strand break. In this article, we provide evidence that this cleavage complex is responsible for inhibition of phage growth and that recombinational repair can reduce sensitivity to the antitumour agent, presumably by eliminating the complex (or some derivative thereof). First, topoisomerase‐deficient mutants were shown to be resistant to m‐AMSA, indicating that m‐AMSA inhibits growth by inducing the cleavage complex rather than by inhibiting enzyme activity. Second, mutations in several phage genes that encode recombination proteins (uvsX, uvsY, 46 and 59) increased the sensitivity of phage T4 to m‐AMSA, strongly suggesting that recombination participates in the repair of topoisomerase‐mediated damage. Third, m‐AMSA stimulated recombination in phage‐infected bacterial cells, as would be expected from the recombinational repair of DNA damage. Finally, m‐AMSA induced the production of cleavage complexes involving the T4 topoisomerase within phage‐infected cells.