Project Summary/Abstract Reactive oxygen species (ROS) generated by exposure to environmental toxins are a constant source of DNA damage. If not properly repaired, this DNA damage can result in subsequent mutations and genomic instability, ultimately resulting in disease. The primary pathway used by cells to repair oxidative DNA damage arising from this environmental exposure is the base excision repair (BER) pathway. One of the key enzymes in BER is DNA polymerase beta (pol ꞵ), which is responsible for the insertion of a nucleotide at the site of a one-nucleotide gap. How pol ꞵ carries out this activity has been well studied using linear DNA. However, most eukaryotic DNA is organized into chromatin, where DNA is extensively bound by nucleosomes. This creates a barrier to efficient BER due to DNA occlusion by the histone octamer. Therefore, repair in this setting is both more difficult and is poorly understood at the mechanistic level. Within nucleosomes, x-ray repair cross-complementing 1 (XRCC1), a non-enzymatic scaffolding protein, has been shown to increase the rate of DNA repair through BER. XRCC1 forms a strong complex with pol ꞵ, with some evidence showing XRCC1 facilitates pol ꞵ recruitment to DNA damage in nucleosomes. However, the mechanism by which XRCC1 fulfills this role with pol ꞵ is unclear. With more than 10,000 oxidative DNA lesions generated per cell per day, and the ubiquitous nature of this damage in chromatin, understanding how BER repairs these lesions in nucleosomes is critical to our understanding of DNA damage repair following exposure to environmental toxins. The goal of this study is to establish how XRCC1 facilitates pol ꞵ DNA damage search, recognition, and repair within chromatin at the structural and mechanistic level. To test this, I propose the following aims: (1) Determine how pol ꞵ and XRCC1 search DNA and recognize DNA damage in chromatin and (2) Determine the structural basis of pol ꞵ and XRCC1 DNA damage recognition in chromatin. To accomplish these aims, I will comprehensively study the mechanisms pol ꞵ and XRCC1 employ to locate DNA damage and what structural changes are induced by the XRCC1/pol ꞵ complex binding DNA damage on nucleosomes. Specifically, correlative-optical tweezers and fluorescence microscopy (CTFM) will determine the mechanisms that pol ꞵ and XRCC1 use to search DNA (Aim 1A) and recognize DNA damage (Aim 1B). Cryo-EM will be used to determine the structure of XRCC1 and the pol ꞵ/XRCC1 complex on nucleosomes containing DNA damage (Aim 2A). Finally, a combination of single-turnover kinetics and gel shift assays will determine the impact XRCC1 has on pol ꞵ single-nucleotide insertion and binding affinity on the nucleosome (Aim 2B). This innovative project will be completed at the University of Kansas Medical Center under the supervision of an expert sponsorship team. In addition to technical skills, the training plan also prioritizes scientific communication, writing, and mentorship. The prop...