Dale Wigley

Molecular enzymology of DNA replication and repair

The replication and repair of DNA requires a vast array of different proteins. Although the overall biological mechanisms involved in these processes are beginning to become clearer, the molecular mechanisms of most of the proteins are still very poorly understood. In order to understand what goes wrong when DNA is damaged or replicated incorrectly, we have been studying a number of systems to learn more about the details of these processes at the molecular level. There are opportunities for both biochemists and crystallographers.

Pre-replication complex - initiation of replication
Initiation of DNA replication in eukaryotes requires assembly of the pre-replication complex. It has been discovered that DNA replication and repair mechanisms in the archeabacteria share many similarities with eukaryotes. We are therefore investigating the pre-replicative complex in these organisms as a simplified model for the human complex. We have cloned and expressed the origin recognition proteins (ORC1 and ORC2) and the MCM helicase that together comprise the core of the pre-replication complex. A biochemical investigation of these proteins is underway with a view, eventually, to structural studies of the entire complex bound to a replication origin. The crystal structureof ORC2 has been obtained. We have also been carrying out biochemical studies and have managed to assemble a replication origin in vitro. Biochemical and structural work will be continuing on this project.

RecBCD - a key component in recombination repair in E.coli
The RecBCD protein is a key player in recombination repair of double strand DNA breaks. Using a comnination of two helicase subunits and a mutli-functional nuclease, the enzyme complex processes the blunt ends and loads them with RecA protein at Chi sites. We have recently determined the crystal structure of RecBCD complexed with DNA and are now beginning to undertake biochemical studies based on this structure in order to understand more about how the enzyme carries out its varied tasks.

Replication factor C - loading of polymerase at the fork
Many processive enzymes that operate on DNA form rings that encircle the DNA, creating a topological problem. We are interested in understanding how these rings are loaded onto DNA. To this end we have begun to study Replication Factor C (RFC), a protein complex comprising two sorts of protein subunit that forms rings. These rings function to load the processivity factor of DNA polymerase (called PCNA) onto DNA. In eukaryotes, there are five different, but related, proteins that together comprise heteropentameric RFC.

By contrast, we have shown that the archaeal RFC forms heteropentameric rings with one large and four copies of identical small subunits. We have also shown that loading requires ATP binding but not hydrolysis and only involves a subset of the possible ATP-binding sites within the complex. Using site-directed mutants we have analysed which subunits bind ATP and we are using this information to assist in efforts to crystallise an intact RFC/PCNA/DNA complex for structural studies.

References

1. Singleton MR, et al. Nature 2004; 99: 13492-13497.
2. Seybert A and Wigley DB. EMBO J 2004; 23: 4579-4583.
3. Singleton MR, Scaife S and Wigley DB. Cell 2001; 107: 79-89.
4. Dillingham MS, et al. Proc Natl Acad Sci USA 2001; 98: 8381-8387.
5. Soultanas P, et al. EMBO J 2000; 19: 3799-3810.