This project is designed to develop a novel proteomic approach for detecting and identifying transient protein-protein associations formed during severe metabolic stress. It will address specifically the protein interactions required for translesion DNA synthesis and inducible responses that are initiated by stalled replication complexes. The investigators plan to demonstrate the efficacy of this approach by using it to identify proteins that bind to DNA polymerase eta both before and during DNA replication and/or translesion synthesis. Pol eta plays an important role in mutation avoidance by catalyzing efficient and accurate replication past UV-induced cyclobutane pyrimidine dimers. Absence of such activity underlies the cancer-prone syndrome xeroderma pigmentosum variant. Although this is a rare autosomal recessive disease, mutations in other gene products that regulate or interact with pol eta might also increase cancer risk. The binding partners of pol eta could represent functional components of protein complexes that catalyze translesion synthesis, and/or negative regulators that block access of this bypass polymerase to undamaged DNA, which is replicated by higher fidelity DNA polymerases alpha and delta. Recombinant pol eta, tagged with six histidines and a c-myc epitope, will be modified with chemical groups that can be photo-activated to cross-link binding proteins. The investigators will demonstrate that the modified pol eta retains DNA polymerization and lesion bypass activities by using primer extension assays (single enzyme catalysis)and SV40 origin-dependent in vitro replication of closed circular duplexes (requiring interaction of pol eta with other replication factors). Pol eta partners will be identified by mass spectrometry of tryptic fragments of the proteins covalently bound to it by the photo cross-linking reaction. Once the proteins making direct contact with pol eta are identified, these primary partners can be used to search for other proteins within the translesion synthesis complexes and/or the network of signal sensors and transducers responding to stalled replication complex. Starting with pol eta as the initial bait, this project has the potential to develop into a major research initiative leading to the characterization of modules of proteins involved in post-replication repair and other DNA damage responses.
