The faithful and efficient transcription of genomic DNA into mRNA is crucial for cell survival under DNA damage caused by UV irradiation, oxidative stress, or chemical DNA modifications. To maintain genomic integrity, cells have evolved separate cellular strategies involving multiple DNA damage repair and DNA damage tolerance mechanisms. Non-bulky DNA lesions are preferentially repaired by the base excision repair (BER) pathway while damages that cause large DNA distortion, such as UV light-induced cyclobutane pyrimidine dimers (CPDs)/cisplatin adducts and oxidative cyclopurines are primarily subject to the nucleotide excision repair (NER) pathway. Despite ongoing repair, some lesions escape detection, presenting the cell with a challenge for continued DNA and RNA synthesis. During replication, the deleterious effect of DNA lesions can be alleviated by translesion DNA synthesis (TLS). During TLS, the high-fidelity replicative DNA polymerases are switched transiently to specialized translesion DNA polymerases that can accommodate bulky lesions within a more spacious active site, thus enabling their bypass. In the past 4 years our work on transcription-coupled DNA repair (TCR) revealed that yeast Pol II has an intrinsic capability to bypass UV-induced CPDs as an alternative pathway to its preferential repair by TC-NER. We defined this process as RNA polymerase translesion synthesis (RTLS, here and herein) to distinguish it from the regular TLS by DNA polymerases. We further demonstrated that the efficiency of the CPD bypass by Pol II correlates with increased UV cell resistance. Most importantly, our most recent unpublished results revealed that mammalian Pol II employs the similar mechanism to negotiate with the CPDs in vitro. To address the functional correlation between RTLS and TC-NER, we will investigate the mechanism for strand specific repair in various genetic backgrounds in yeast as a model system. We will expand Project 2 to transcription across the other types of DNA lesions including cyclopurine lesions (cyclo-dA and cyclo-dG). Our preliminary results showed that yeast and mammalian Pol II are capable of transcription through cyclo-dA in vitro by using a mechanism, which is strikingly similar to transcription across the CPDs. We will continue elucidation of the role of Rpb4/7 and Rpb9 subunits of Pol II in initiation of TCR and will search for repair proteins specific to Rpb4/7- and Rpb9-dependent TCR pathways. In the long term, this project will be merged with """"""""Transcription Fidelity"""""""" project to evaluate an impact of faithful and error-prone transcription to genome integrity during normal growth and endogenous/exogenous stresses caused by DNA damage, tumorigenesis and aging.
