This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our objective is to determine the three-dimensional structure of the proliferating cell nuclear antigen (PCNA) in complex with a domain from the DNA polymerase Rev1. Rev1, a member of the translesion synthesis DNA polymerases, allows replication past DNA lesions, making it a key factor in DNA damage-induced mutagenesis in eukaryotes. Recent studies have shown that the high processivity of Rev1 at DNA lesions necessitates its interaction with PCNA. It was also shown that this interaction is stabilized during the DNA damage response. In agreement with these studies, our in vitro binding assays using calorimetry and NMR spectroscopy have demonstrated a tight interaction between PCNA and a BRCT domain located at the N-terminus Rev1. We have obtained crystals of the PCNA/Rev1-BRCT complex that are suitable for structure determination. PCNA was enriched in selenomethionine to collect MAD or SAD data.
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