Project Summary – Maintaining active high-fidelity replication despite a constant barrage of DNA damage or blocks that are encountered by the replisome requires several inherent genomic protective functions. An immediate first ‘on-the-fly’ response can be the recruitment of translesion synthesis (TLS) DNA polymerase to directly bypass damage that stalls a high-fidelity (HiFi) polymerase. However, this process is complex and multidimensional and requires separate substitution events that can recruit one of several TLS polymerases, facilitate insertion opposite the lesion and beyond, and then substitute back to the HiFi polymerase. Although TLS polymerases have generally accurate insertions opposite cognate lesions, their fidelity opposite undamaged DNA must be restrained to prevent downstream mutations giving rise to cancer initiation. The kinetic and structural mechanisms for TLS insertions have been widely studied, providing a wealth of information on lesion specificities; however, these results are primarily derived from truncated core polymerase enzymes that lack N- and C-terminal domains important for interacting with replisome components to facilitate TLS substitutions. Moreover, the molecular and structural mechanisms to limit downstream synthesis after insertion by TLS polymerases are unexplored. We hypothesize that intrinsic contacts outside the active site of TLS DNA polymerases restrict synthesis downstream of a lesion to maintain genomic fidelity. To test this hypothesis, we will validate our preliminary data showing that specific ‘pink-trigger’ residues in TLS polymerases sense synthesis at distinct positions past lesions to kinetically promote dissociation. Utilizing primarily full-length human DNA polymerase enzymes, accessory factors, and stabilizing bridges, we will characterize the substitution steps needed to bypass several cognate lesions, validating important contact points that help recruit, synthesize past, and enable reinstatement of HiFi DNA polymerases. The proposed research program is highly integrated using advanced biochemical, enzymological, kinetic, and structural approaches to better understand the entire TLS process and will be performed by several excellent undergraduate and graduate student researchers providing a significant health science training opportunity at a primarily undergraduate university. Results from this proposal will provide a clearer understanding of the steps and contacts required to perform efficient TLS but also restrict downstream low-fidelity synthesis past template lesions. Conclusions from these studies will be influential in providing insights into patient mutations leading to cancer and rapid aging as well as identifying novel targets to prevent chemoresistance.