Deciphering the progression and regulation of human translesion DNA synthesis

Project Summary/Abstract The overarching goal of my research program is to understand how the human genome is faithfully replicated when it is constantly damaged by covalent modifications that can alter its coding properties. A focus is on translesion DNA synthesis (TLS), the predominant DNA damage tolerance (DDT) pathway utilized in humans to replicate damaged DNA. TLS utilizes specialized DNA polymerases that can accommodate an array of DNA damages, albeit with lowered fidelities, and promotes cell survival in the face of DNA damage by allowing replication of a damaged genome to continue. However, tight regulation is critical as aberrant TLS can selectively propagate cells with increased mutagenesis and chromosomal rearrangements, which can lead to cancer. Furthermore, because TLS promotes cell survival after exposure to DNA damaging agents, aberrant TLS can also afford cancer cells the ability to overcome common chemotherapies that aim to trigger cell death by acutely damaging DNA. Hence, TLS is a promising candidate for targeted cancer co-therapy. Despite the established links between human health and TLS, the progression and regulation of TLS is unclear and many key gaps persist in our fundamental knowledge that cloud our understanding of the contribution of TLS to genetic inheritance and carcinogenesis. For example, human DDT can occur by at least three pathways but what is the interplay between TLS and other DDT pathways? My long-standing interests and vast expertise in human DNA damage repair and replication and my established ability to utilize a multi-faceted approach integrating molecular biology, biochemistry, and biophysics provide a unique opportunity to fill these gaps. This proposal will address the progression and regulation of TLS by tackling two broad areas that are each a cornerstone of our fundamental understanding of TLS and DDT in general; 1) Activation of TLS and; 2) The interplay between DDT pathways. To do so, we design and apply unique, quantitative approaches that utilize kinetic techniques and can be adapted to many biological scenarios. The PCNA sliding clamp is an essential DNA replication factor and is monoubiquitinated at sites of DNA damage by the Rad6(Rad18)2 complex. PCNA monoubiquitination is imperative for human TLS and activates this critical pathway by recruiting TLS factors. Area 1 will investigate how the activity of the human Rad6(Rad18)2 complex is regulated to efficiently monoubiquitinate PCNA at DNA damage sites, activating TLS. Human DDT can occur by at least three pathways including TLS and all emanate from a common intermediate. Area 2 will investigate the interplay between TLS and other DDT pathways and decipher functional relationships between key events in each pathway. This proposal investigates TLS in broad contexts that consider the complexities and dynamics of cellular environments and will significantly advance our fundamental understanding of how the human genome is faithfully replicated in the face of DNA damage and inspire new avenues of research in cancer etiology and treatment.