Multiple strategies have evolved to minimize the genotoxic consequences of endogenous and environmental agents that damage DNA. The ubiquitous process of excision repair removes a variety of structurally unrelated DNA lesions that are mutagenic and that may result in malignancy, faulty differentiation patterns and cell death. The efficiency of excision repair varies throughout the genome and a dedicated pathway of transcription-coupled repair (TCR) deals with lesions in the transcribed strands of expressed genes. Our favored model for the mechanism of TCR implicates an arrested RNA polymerase as a signal to recruit excision repair enzymes to the transcription blocking lesion. To understand how repair proteins recognize an arrested RNA polymerase to initiate a repair event, we intend to continue characterizing the unique features of the transcription complex when it encounters different impediments during the normal course of elongation. We propose to extend this analysis to elucidate the protein-interactions of the RNA polymerase complex when it is arrested at a cyclobutane pyrimidine dimer (CPD). Utilizing an in vitro transcription assay with purified T7 RNA polymerase or rat liver RNA polymerase II (RNAP II) and unique DNA substrates we will analyze the behavior of RNA polymerase at site-specific lesions, focusing upon lesions that impose different types of constraints, including an abasic site or oxidized abasic site; a DNA bubble structure (intermediate in TCR); psoralen monoadducts and interstrand crosslinks; and mismatch repair complexes bound to 8-oxoG (a lesion that does not in itself arrest RNAP II). Protein-interactions and protein-modifications of RNAP II transcription complexes when arrested at a DNA lesion will be identified and characterized. Isolated RNAP II ternary complexes will be incubated with relevant purified proteins (e.g. XAB2, CSA, CSB, TFIIH, XPG, MSH2-MSH6, MSH2-MSH3) in the presence or absence of the elongation factor SII to determine by gel mobility shift assays how coupling factors interact with an arrested polymerase. The position of the transcription """"""""bubble"""""""" following RNAP II arrest at a DNA lesion will be mapped with KMnO4, before or after addition of putative coupling factors; and after SII-mediated reverse translocation of RNAP I1. To study the role of ubiquitination upon transcription arrest at a lesion, the isolated RNAP II ternary complexes will be incubated with HeLa cell extracts and His-tagged ubiquitin, followed by detection of the reaction products by Western blotting. The unique features of a single translocating RNAP II complex as it is arrested at a CPD, and reversed by SII will be visualized using an optical trap. Ultimately our goal is to reconstitute TCR in vitro, building upon and benefiting from results obtained as we achieve the foregoing aims. This research is relevant to an understanding of the relationship between DNA repair and oncogenesis, as well as the possible application of such understanding to more effective cancer therapy.
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