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DNA Replication, Recombination and Repair.

DNA replication, recombination, and repair are fundamental processes essential to the perpetuation of the genome carried out by large protein machines. There is a growing consensus that these processes are integrally related and that they function by similar mechanisms. Replication protein A (RPA), a heterotrimeric protein of 70, 32 and 14 kDa subunits, represents one of the primary direct linkages between DNA replication, recombination and repair, since it's presence is absolutely required for each of these processes. RPA is the major single-strand DNA binding protein in eukaryotes, but has been shown to also mediate numerous interactions with other essential proteins in each of the corresponding protein assemblies. Our research is directed to localizing, quantitating and structurally describing the interactions of RPA with its DNA and protein substrates. These results will clarify the biochemical functions of RPA and establish the role of RPA in linking DNA replication, recombination and repair.

We produce intact RPA heterotrimer and a variety of smaller structural domains and fragments and use these to map binding to single-strand DNA and target proteins. A combination of limited proteolysis, affinity chromatography, and mass spectrometry is used to identify the structural domains involved in these interactions. The strength of the interaction is then determined by biophysical methods such as fluorescence, surface plasmon resonance, and NMR spectroscopy. To build up a molecular view of DNA replication, recombination and repair machines, we apply the tools of high resolution structural biology (NMR spectroscopy, X-ray crystallography, computation) to determine the three-dimensional structures of complexes of RPA domains, DNA and target proteins.

Current Projects involve determining the roles of RPA in mediating:

1. Nucleotide excision repair of damaged DNA. Work completed on damage recognition protein XPA, and current effort focused on XPF/ERCC1 and XPG excision nucleases.
2. Replication. Substantial progress made on the T-antigen initiator protein from the SV40 model system, preliminary results obtained on DNA polymerase/primase. Work originating on RecQ helicases associated with Bloom and Werner's Syndrome.
3. Recombination. Physical mapping and sub-cloning of structural domains underway for RAD51.

Signal Transduction by EF-hand Calcium-Binding Proteins | DNA Replication Recombination and Repair