PROJECT SUMMARY Cells have evolved complex machinery for both the replication of DNA and for repairing errors in DNA. Herpes Simplex Virus 1 (HSV) is a large double strand DNA virus that replicates in the nucleus of the host cell and commandeers some of this host replication machinery. Despite decades of study, the mechanisms of HSV DNA replication are still poorly understood. The incoming genome contains nicks and gaps, and it is not known when or if these are repaired in relation to the timing of DNA synthesis. HSV encodes seven essential DNA replication proteins including an origin binding protein, a single strand DNA (ssDNA) binding protein (ICP8), a three- subunit helicase/primase (UL5/UL8/UL52), and a two-subunit polymerase (UL30/UL42). In addition, HSV also encodes a two-unit recombinase consisting of a 5'-3' exonuclease (UL12) that functions with ICP8. Isolation of proteins on nascent DNA (iPOND) is a powerful tool to study DNA replication because it allows for the specific purification of replication forks away from bulk chromatin. When coupled with SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics, the iPOND-SILAC-MS method provides a robust, unbiased discovery tool to identify fork associated proteins by determining the intensity of proteins in a pulse sample compared to a chase sample. We have utilized iPOND-SILAC-MS to generate robust data sets of the protein composition of HSV replication forks and replication forks lacking UL12. In the absence of UL12 a cellular deubiquitinating enzyme, USP15, is not recruited to replication forks. USP15 interacts directly with UL12 and is required for efficient HSV replication. In addition to viral proteins, many cellular proteins are enriched on viral replication forks. In twelve iPOND-SILAC- MS we have identified 200-300 proteins (viral and host) enriched on HSV DNA replication forks. The overall goal of this research proposal is to identify the host proteins associated with viral DNA to generate a more complete understanding of the mechanisms of HSV DNA replication. To this end we will address three overlapping research areas in my laboratory: 1) Characterize the physical interaction of UL12 with USP15, 2) Identify the human replisome proteins required for HSV DNA replication, 3) Determine the fate of the nicks and gaps in the incoming viral genome.

Project Narrative Herpes Simplex Virus types 1 and 2 are ubiquitous pathogens that are widespread in the population and cause a variety of diseases including oral lesions, genital ulcers, and ocular infections that can result in blindness. Our goal is to contribute to the understand of the basic mechanisms of viral DNA replication which will contribute to the design of antiviral therapies.