The Project (Transcription-Coupled and Replication-Associated Excision Repair) focuses on mechanisms coupling DNA excision repair machinery with transcription and replication. BothNucleotide Excision Repair (NER) and Base Excision Repair (BER) are highly coordinated by interactionsbetween proteins in the pathway. Moreover, they are preferentially targeted by specialized transcriptioncoupledrepair (TCR) machinery to lesions that affect transcription elongation or by replication-associatedrepair (RAR) to lesions near the replication fork or in recently replicated DNA. We hypothesize that theseinteractions and their effects on function are regulated through unstructured flexible regions that undergodisorder-to-order transformations upon complex formation and/or post-translational modifications. We willtest this overall hypothesis and specific hypotheses in five Aims by collaborative studies to characterize,validate, and map interactions, identify damage-induced modifications, observe effects of complexes on DNAstructure by scanning force microscopy (SFM), and visualize subunits and complexes by electronmicroscopy (EM), small angle X-ray scattering (SAXS), and protein crystallography (PX).
Aim 1 willstructurally characterize early steps of TCR: recognition by XPG and CSB of RNA Polymerase II (RNAPII)stalled at a lesion, and remodeling of RNAPII by TFIIH to allow access to the lesion. SFM and EM studieswill test the hypothesis that these occur by ordered conformational changes.
Aim 2 will structurallycharacterize CSB and reinvestigate its causal role in CS by determining whether mutant CSB interferes withresponses to oxidative DNA damage through non-productive interactions with other proteins in the pathway.
Aim 3 will investigate the identified interactions that couple BER and NER to transcription through (a) SAXSand PX studies of XPG protein and its domains and complexes, (b) analysis of interactions of NEIL2 withRNAPII, XPG and CSB, and (c) characterization of the effect of post-translational modifications on XPG andNEIL2 interactions.
Aim 4 will characterize the structural basis for BER pathway coordination by interactionsof NEIL1 and NEIL2 glycosylases with downstream BER proteins and test the hypothesis that BER pathwayprogression results in progressive DNA bending.
Aim 5 will investigate molecular mechanisms of RAR bydetermining the structure of the checkpoint sliding clamp -- the 9-1-1 complex - and by characterizinginteractions of the MYH and NEIL1 glycosylases with PCNA and 9-1-1. The anticipated outcome is amolecular understanding of cancer predispositions and developmental disorders that arise from defects inthe coordination of excision repair with transcription and replication. Collaborations of Project 2 within SBDRand with the UCSF Comprehensive Cancer Center will relate results of these studies to genome integrity andcancer etiology as well as to development of promising molecular targets for cancer drug discovery.
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