In addition to providing all the proteins necessary for replication, T4 phage also is able to perform the necessary DNA repair functions for the maintenance of its genome. However, unlike DNA replication, most of the DNA repair pathways in T4 phage have not been fully reconstituted in vitro. To accomplish this goal, we have cloned, expressed, and purified all the T4 proteins thought to be necessary for carrying out double strand break (dsb) repair (figure below). These are briefly described below.

The central protein in recombination-dependent processes in T4 phage is UvsX (UV sensitivity gene X). As its name implies, mutation of UvsX results in phage that are more sensitive to DNA damaging agents such as UV light. UvsX is an ATP-dependent recombinase with sequence homology to E. coli RecA protein. UvsX has been purified and its biochemical properties are well characterized. Additionally, UvsX has been used to reconstitute recombination dependent initiation of replication by our lab and others.

Most organisms that contain a recombinase also encode for a recombinase mediator protein (RMP). The RMP in T4 phage is the UvsY protein. Like UvsX, UvsY has been purified and biochemically characterized. UvsY is necessary for the loading of UvsX onto ssDNA that is coated with ssDNA binding protein (gp32) or under conditions of high ionic strength.

The third protein to make up the UV sensitivity group is UvsW. We recently discovered UvsW contains both helicase and strand pairing activities and is a functional homolog of the RecQ protein. We have proposed several roles for UvsW in putative DNA repair pathways that require both unwinding and strand annealing activities.

The final protein thought to be necessary for recombination dependent DNA repair is gp46/47. This protein may be responsible for the 5’ to 3’ resection of the blunt-ended DNA that results from a dsb. We are actively investigating the biochemical properties of this complex with the goal of defining its biological function and reconstitution of the dsb repair pathway.

In parallel to our work with T4 double strand break repair proteins, we have initiated a study of the eukaryotic gp46/47 homologs, Rad50/Mre11. The precise function of this complex is controversial, with genetic evidence indicating it is the nuclease that performs 5’ to 3’ dsb resection and biochemical evidence demonstrating that its nuclease activity is 3’ to 5’, which is incompatible with its proposed in vivo role. We have cloned, expressed, and purified the proteins from S. cerevisiae. We are in the process of developing an in vitro system to study the effects of other proteins on the function of Rad50/Mre11.