Structural and Mechanistic Studies of DNA Damage Bypass Pathways in Eukaryotes

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Project Summary/Abstract DNA damage is a serious threat to genome stability. This is because it interferes with DNA replication leading to mutations and chromosomal rearrangements – the hallmarks of cancer, aging, and other diseases. To ensure genome stability, cells utilize DNA damage bypass pathways to cope with DNA damage during replication. The long-term goal of our research program is to understand how DNA damage bypass is carried out in eukaryotic systems at the structural and mechanistic level. Our research will focus on two damage bypass pathways: translesion synthesis and template switching. Progress in this field has slowed recently because of the challenges associated with studying how the various bypass components assemble into and function within large, dynamic, multi-protein complexes. We have developed the biochemical, biological, biophysical, computational, and structural tools needed to overcome these challenges. This puts us in a unique position to answer many fundamental questions about damage bypass. Our future research plan is organized into three broad projects. First, we will study the regulation of DNA damage bypass. This will be done by determining how bypass complexes are assembled at stalled replication forks and by determining how this assembly is controlled by PCNA-ubiquitylating enzymes. Second, we will study the mechanisms of translesion synthesis. This will be done by determining how the most appropriate non-classical polymerase is chosen to bypass the damage and by determining how each non-classical polymerase accommodates damaged DNA templates. Third, we will study the mechanisms of template switching. This will be done by determining how the remodeling of the replication fork allows for the bypass of DNA damage and by determining how this process is carried out by fork-remodeling DNA helicases. In answering these questions, we will gain important new insights into the maintenance of genome stability.