PROJECT SUMMARY It is thought that many diseases, including cancer, begin at the DNA level. DNA is constantly being bombarded by endogenous and exogenous factors, with UV rays as one of the most powerful agents. The cell's mechanism of defense is a class of enzymes known as DNA polymerases which repair DNA damage and are regarded as guardians of the genome. Of these DNA polymerases, DNA Polymerase Theta (POLQ) has been shown to repair UV damage including the common lesions cis-syn cyclobutane–pyrimidine dimer (CPD) and a (6-4) photoproduct (PP). Despite this role, POLQ introduces mutations during this type of repair, and it is unclear if it protects the genome or contributes to genomic instability. The objective of this project is to determine the biochemical and mechanistic basis for how POLQ repairs UV-induced DNA damage through a variety of biochemical, biophysical, and cellular biology approaches. This proposal will test two hypotheses about how cancer- associated variants of POLQ in patients from sun-exposed tumors bypass CPD-damaged DNA. The knowledge gained from these experiments on the biochemical mechanisms needed to bypass UV-damage will add supporting evidence to the idea that POLQ protects again skin cancer in normal cells despite low levels of mutagenesis and that the cancer-associated variants are drivers of tumorigenesis. This is a first-of-its-kind study that explores the biochemical mechanism and cellular phenotypes of patient-derived mutants of POLQ. The long-term scientific goal of the Towle-Weicksel research group is to fully investigate how structure and function of DNA polymerases influence genomic stability and carcinogenesis. To achieve these goals, this proposal will determine the catalytic mechanism of patient-derived variants of POLQ (Aim 1) and to determine the mechanism of nucleotide selection of POLQ (Aim 2). Preliminary studies using biochemical kinetics and other cellular biology techniques suggest that one of the cancer-associated variants experiences altered DNA repair abilities and is sensitive to DNA damaging agents. These inherent differences between the variant and wild-type POLQ provide an opportunity to explore the biochemical mechanism further to expand our current understanding of how the cell copes with UV-induced damage. Undergraduate researchers from Rhode Island College will play an integral role in the success of this R15 AREA proposal. They will be involved in all aspects of the project including experimental design, data interpretation, and manuscript preparation to foster an undergraduate research experience that advances scientific discovery and cultivates scientific skills.