Herpes simplex virus type 1 (HSV-1) is a prevalent pathogen that infects the majority of the human population. Much of the virus life cycle occurs in the nucleus of infected cells, where events that occur on the viral genome determine the outcome of infection. However, how the structure of the viral DNA contributes to the regulation of key viral processes is not well understood. Virion HSV-1 DNA enters into host cells containing single strand breaks and single stranded gaps. During viral DNA replication, branched genome structures form, likely as recombination-mediated replication intermediates. We previously demonstrated that distinct groups of cellular DNA repair proteins associate with viral genomes early during infection and after the onset of viral DNA replication. How DNA damage is navigated by viral DNA replication, transcription, and packaging machinery is not understood. Furthermore, how the genome-wide structure of the HSV-1 genome is modified during the temporal progression of infection has not been defined. A major roadblock to previously investigate these questions was the inability to probe genome-wide DNA structure. In this exploratory proposal, we describe our plan to develop approaches to define the structure of the HSV-1 genome throughout productive infection. The aims outlined in this proposal will establish new approaches to map single and double strand breaks on HSV-1 DNA and investigate viral replication and recombination intermediates in vivo. Results will provide greater insight into mechanisms of regulation of HSV-1 transcription, DNA replication, recombination, and repair. Furthermore, tools generated could be adapted to probe HSV-1 genomes before and after reactivation, viral infection in cells deficient for select cellular DNA repair and DNA damage response factors, and genomes of other DNA viruses.