CO CRYSTALS OF HUMAN REPLICATION PROTEIN A & SINGLE STRANDED DNA

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Replication protein A (RPA) is a key component of the DNA replication and repair machineries. RPA is a single stranded DNA binding protein that interacts with DNA replication orgin binding proteins, DNA polymerases, transcription activator proteins and DNA repair factors, including those that are mutated in several forms of human cancer. RPA is a heterotrimer; the DNA binding domain has been localized to the largest subunit. We have used limited proteolysis and deletion analysis to identify the minimal single strand DNA binding domain. We have cryslalized this domain together with ssDNA. Cryo-protected crystals diffract to a resolution of 2.2A on our RAXIS-II equipped with double double focussing mirrors. We are pursuing heavy atom searches on our unit and have prepared selenomethionine-labelled protein for co-crystallization. In this proposal, we wish to use, collect higher resolution native as well as heavy atom data sets.