Project Summary: Eukaryotic cells face constant challenges to the integrity of their genome from both endogenous and environmental sources, and sophisticated processes have evolved to respond to and minimize long-term DNA damage. Cells employ DNA damage tolerance (DDT) pathways to ensure the completion of DNA replication by filling in gaps created by replication barriers or lesions in the DNA. By allowing replication to continue even in the face of minor lesions, DDT pathways prevent replication forks from stalling, which can lead to fork collapse resulting in serious chromosomal abnormalities and genome instability. Nevertheless, at least one arm of the DNA damage tolerance pathway, known as translesion synthesis (TLS), can be mutagenic, the extent to which depends on the lesion and TLS polymerase involved. Thus, considered broadly, DNA damage tolerance pathways help maintain genome stability but can also promote mutagenesis, which can contribute to cancer and drug resistance. The overall goal of this proposal is to understand the molecular mechanisms that govern damage-specific lesion bypass. In the previous funding period, we identified SHPRH and HLTF as multi-functional proteins that contribute to multiple aspects of DDT pathways in mammalian cells. These proteins exert their effects in a damage-specific manner, responding to alkylation damage and UV damage with complementary specificities, yet the mechanisms by which they act remain undefined. In order to understand how SHPRH and HLTF regulate DNA damage tolerance, we will study their regulation and their role in DDT using a combination of genetic and biochemical approaches. The first aim of this proposal will address how HLTF and SHPRH are regulated following DNA damage. We will analyze the interaction of HLTF and SHPRH with damaged replication forks, and determine how different types of DNA damage affect the interaction of HLTF and SHPRH with Rad18, another regulator of DDT. The second aim will address the function and mechanism of action of HLTF and SHPRH in DDT. We will ask how these proteins affect mutagenesis and template switching, the protein composition of stalled forks, and the stability of repeat sequences. Collectively, these studies will provide novel insights into the regulation of two critical regulatory genes that help to minimize mutations arising from replication stress. Both HLTF and SHPRH have been found to be altered in numerous cancers, hence these studies are highly relevant to cancer and other diseases that are modified by genetic mutations.