DNA damage tolerance strategies at the organismal level

About This Grant

Project Summary The focus of my lab is to understand the mechanisms of genome instability caused by error-prone DNA repair and damage tolerance mechanisms. Specifically, we are interested in the roles of translesion DNA polymerases in double-strand break repair and lesion bypass. Recent studies have shown that cancer cells upregulate the expression of translesion polymerases and that they can become addicted to them, providing potential therapeutic targets. However, because most of these studies utilize cells grown in culture, tissue- and development-specific context that may be important for understanding treatment efficacy is often lost. To address this, we have pioneered the use of a Drosophila model to study DNA damage tolerance and error- prone double-strand break repair pathways. We made the important discovery that DNA polymerase theta is a key protein involved in alternative end-joining repair of double-strand breaks, which prevents the creation of large deletions when homologous recombination repair is impaired. We recently demonstrated that highly proliferative tissues in Drosophila rely heavily on translesion synthesis (TLS) bypass to complete replication and prevent genomic catastrophe following alkylation damage. We identified at least two ways that the REV1 translesion polymerase scaffold protein promotes bypass of alkylation damage, and we have shown that the SLX1/4 structure-specific nuclease becomes important for tolerance in a TLS-deficient background. Using a forward genetic screen for mutations that impair survival specifically when TLS is compromised, we have identified several novel genes that promote tolerance of both exogenous and endogenous DNA damage. Building on these studies, we plan to advance our research program in several directions. First, we will further determine the contribution of template switching and fork reversal mechanisms to damage tolerance in Drosophila, as these represent critical backup pathways that could contribute to therapeutic resistance when TLS is impaired. Second, we will use genetic, molecular, and biochemical assays to precisely define how the genes identified from our screen act to promote replication past DNA lesions, prioritizing those that have not previously been associated with damage tolerance. Third, we will investigate the mechanistic basis behind our observation that haploinsufficiency of the BRCA2 gene causes synthetic lethality when key replication and repair proteins are mutated, focusing on bypass of endogenous DNA damage. We will utilize chromosome spreads, damage visualization techniques, and whole-genome sequencing to determine the consequences of loss of key tolerance mechanisms on genome integrity. Together, these studies will provide important insight into the ways that cells prioritize various damage tolerance and repair strategies in the context of developing tissues, and the consequences that these strategies have on mutagenesis and chromosome instability.