Emerging Mechanisms of Replication-coupled DNA Repair

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Cells are constantly exposed to exogenous and endogenous agents that chemically damage the genome. During DNA replication, this chemical DNA damage can introduce mutations, chromosomal rearrangements, and chromosome mis-segregation events that contribute to progression of cancer and ageing. DNA interstrand cross- links (ICLs) are highly toxic DNA lesions that covalently link the two strands of DNA and block unwinding by the replicative CDC45/MCM2-7/GINS (CMG) helicase. These lesions are generated by cancer chemotherapeutics, endogenous metabolites, and microbiome toxins. Deficits in ICL repair cause the bone marrow failure and cancer predisposition syndrome Fanconi anemia (FA). The products of genes implicated in FA participate in a common ICL repair pathway that is activated when CMG collides with an ICL. Replication fork stalling at the ICL initiates nucleolytic incisions that convert the ICL into a DNA double strand break (DSB). The DSB is itself a potential source of genome instability that must be repaired by homologous recombination. In previous work, we used Xenopus egg extracts to demonstrate that certain ICLs are repaired by an alternative pathway that is also activated upon CMG collision with an ICL. In this pathway, the NEIL3 glycosylase cleaves an N-glycosyl bond in the cross-link, resolving the ICL without DSB formation but generating a labile abasic (AP) site. Our work indicated that the NEIL3 pathway is the preferred response for resolving a subset of ICLs, though the FA pathway can process these lesions when NEIL3 is inactivated. We further showed that the AP site produced by NEIL3 forms a DNA-protein cross-link with the HMCES protein, which stabilizes the AP site and regulates mutagenic DNA synthesis past the AP site. These results indicate that multiple functionally distinct pathways can cooperate to promote efficient replication-coupled repair of DNA damage. In this proposal we will use approaches spanning biochemistry, molecular biology, and cell biology to investigate how repair mechanisms are coordinated at the replication fork during repair of physiologically- and clinically-relevant DNA lesions. In Aim 1, we will determine the mechanisms of repair for an ICL formed by a bacteria toxin implicated in cancer progression, providing new insight as to how the chemical structure of an ICL influences repair. In Aim 2, we will explore the repair of ICLs by the NEIL3/HMCES pathway, including examining how this pathway is activated and how it regulates ICL repair outcomes. In Aim 3, we will examine how HMCES regulates AP site metabolism and contributes to genome stability in cells. These experiments will provide a deeper understanding of how different biochemical repair activities are integrated at stalled replication forks. This work has the potential to inform therapeutic interventions that modulate replication-coupled repair to sensitize cancer cells to chemotherapy or halt progression of diseases caused by DNA repair deficiencies.