Novel Mechanisms through which translesion synthesis protects genome integrity

Exposure to ubiquitous environmental carcinogens, such as polycyclic aromatic hydrocarbons and UV light, is a major cause of human disease. It is well accepted that genetic mutations are an important step in the development of cancer. It has become clear that such mutations are introduced in part by error-prone DNA polymerases. In response to many environmental genotoxins, eukaryotic cells have evolved alternative methods of replicating damaged DNA via the Translesion DNA synthesis (TLS) Polymerases, consisting of DNA Polymerase eta (Polη), DNA Polymerase kappa (Polκ), DNA Polymerase iota (Polι), and Rev1. TLS is a DNA damage tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome Xeroderma Pigmentosum Variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates PCNA, but the basis for recruitment of Rad18 to PCNA is poorly understood. This dissertation studies several aspects of regulatory mechanisms that contribute to the damage-induced activation of Rad18 E3 ligase activity at PCNA. First, we report a novel role for Polη, the XPV gene product that is mutated in XPV, in targeting Rad18 to PCNA to initiate TLS. Using structure-function analyses and immunofluorescence microscopy, we identified a C-terminal domain of Polη that physically bridges Rad18 and PCNA to facilitate redistribution of Rad18 to stalled replication forks and promote PCNA monoubiquitination. This scaffold function is unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length, catalytically inactive Polη exhibit increased recruitment of error-prone TLS Polymerases after UV irradiation, indicating that maintaining the bridging function of Polη in the absence of its catalytic activity greatly predisposes to mutagenesis. These findings define a molecular basis for TLS pathway activation and provide a new mechanism for mutagenesis and genomic instability in XPV individuals. Next, this dissertation reports novel mechanisms of regulating TLS via stress-activated protein kinase (SAPK) phosphorylation of Rad18 and via Chk1-dependent phosphorylation events. Finally, this dissertation presents data indicating that TLS is involved in the tolerance of oncogene-induced replication stresses and potentially oncogene-induced mutagenesis.Doctor of Philosoph