Project Summary/Abstract Accurate and timely DNA replication is critical for maintaining genomic integrity. Replication forks frequently encounter obstacles such as DNA lesions caused by environmental and endogenous genotoxic agents, conflicts with transcriptional machinery, and unusual DNA structures that block the replicative DNA polymerases leading to fork stalling. There are a large number of proteins that facilitate the resolution of stalled forks. Recently, our lab identified 5-hydroxymethylcytosine binding ES cell-specific (HMCES), or C3orf37, as a protein enriched at replication forks. HMCES contains one SOS Response Associated Protein (SRAP) domain that is evolutionarily conserved through bacteria. A comparative genomics study found that the bacterial HMCES gene is close to SOS operons in bacteria suggesting a function in responding to DNA damage. However, to date, there have not been any functional studies of HMCES. Our preliminary data shows that HMCES-deficient human cells have slow replication kinetics, increased sensitivity to genotoxic agents, and a mutator phenotype. HMCES is recruited to replication forks and UV-damaged chromatin. HMCES binds DNA and interacts directly with components of the replisome. To determine the function of HMCES in genome maintenance, I will define how HMCES binds DNA and HMCES localization to replication forks and UV damaged chromatin using mutagenesis, biochemical, and structural approaches. I will use genetic approaches to test whether HMCES directly acts at replication forks to facilitate accurate completion of DNA synthesis.