Rogers, Colette2024-01-192024-01-192022-11https://hdl.handle.net/11299/260141University of Minnesota Ph.D. dissertation. November 2022. Major: Biochemistry, Molecular Bio, and Biophysics. Advisors: Anja-Katrin Bielinsky, Eric Hendrickson. 1 computer file (PDF); x, 127 pages.Genomic integrity is frequently threatened by DNA damage, such as via exposure to ultraviolet (UV) light or chemotherapeutic drugs, which blocks ongoing DNA replication. Prolonged replication fork stalling can lead to the formation of DNA double-stranded breaks (DSBs), increasing the risk of cancer-causing chromosomal rearrangements or p53-mediated cell death. My thesis research focused on deepening our understanding of the finely-tuned DNA damage response (DDR) pathways that promoted tolerance and repair of replication-blocking lesions. Specifically, by functionally inactivating key DDR factors via CRISPR/Cas9, I took a reverse genetic approach to investigate the function and consequences of several DDR pathways.Chapter 2 of this dissertation is focused on the role RAD18 in regulating DNA damage tolerance (DDT) sub-pathway choice. RAD18 is an E3 ubiquitin ligase that promotes monoubiquitination of the replicative sliding clamp proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) and consequently activation of translesion synthesis (TLS), a mutagenic DDT sub-pathway that allows for replication directly opposite of and past bulky DNA lesions. We confirmed that human colorectal cancer HCT116 RAD18-/- cells are deficient in UV-induced PCNA monoubiquitiantion and TLS activity and discovered that these cells are hyper-recombinogenic (“hyper-rec”). In contrast, neither noncancerous hTERT-RPE1 nor colorectal cancer DLD1 analogous RAD18-/- cell lines exhibited hyper-rec phenotypes. Notably, HCT116 cells have stronger p53 signaling compared to hTERT-RPE1 and DLD1 cell lines. This suggests that the hyper-rec status of some RAD18-deficient cell lines is dependent on their p53 pathway function. Chapter 3 of this dissertation presents substantial genetic data attributing Fanconi anemia (FA)-associated radial chromosome fusions (“radials”) to POLθ-mediated alternative end-joining (A-EJ) activity. FA is a rare genetic cancer-predisposition disorder characterized by defective repair of replication-blocking interstrand crosslinks (ICLs), resulting erroneously in radials. In FA-mediated ICL repair, there is a DSB intermediate that is repaired by homologous recombination (HR). However, in FA pathway-defective cells, the DSB intermediates are repaired by alternative repair pathways, namely classical nonhomologous end-joining (C-NHEJ) and A-EJ. Importantly, we demonstrate that the ICL-induced radials observed in FANCD2-/- and RAD18-/- cells are dependent on the A-EJ factors POLθ and DNA ligase III (LIG3) and occur independently of a functional C-NHEJ pathway. Dysregulation of DDT, the FA pathway, and DSB repair is closely tied to genomic instability and cancer genome evolution. The studies presented in this dissertation are ongoing in hopes of better understanding how the regulation of these DDR pathways can be harnessed for effective anti-cancer therapies. For instance, our laboratory is interested in how damage-induced p53 signaling affects recombination in RAD18-deficient human cells. Furthermore, we are gearing up to sequence, for the first time ever, radial breakpoints in FA cells. We call this project “Radial-Seq”. In addition to the genetic data presented in Chapter 3, Radial-Seq analyses will provide us with detailed information on the molecular make-up of cancer-associated radials.enThesis or Dissertation