PROJECT SUMMARY The overall goal of NIGMS-funded research in my lab is to describe the molecular and cellular mechanisms by which cells sense the presence of DNA damage and how they repair chromosomal double-strand breaks (DSBs). Primarily using budding yeast as a model system, it is possible to induce site-specific DSBs with a high degree of synchrony that is not generally possible in mammalian cells, allowing “in vivo biochemistry” approaches to monitor intermediate steps in DSB repair and DNA damage signaling. We also wish to apply our understanding of DNA repair and recombination mechanisms to elucidate how the Lyme disease bacterium, Borrelia burgdorferi, is able to “change its coat” by repeated gene conversion events. The goals for the next five years of this project focus on understanding how homologous donor sequences are found and used to repair a DSB and how mismatches are tolerated and repaired during different steps of recombination. A second objective is to understand the basis of the 1000- fold increase in mutations associated with DSB repair and how microhomologies are used in repair-dependent template switching, creating complex chromosome rearrangements analogous to events recently found in human cancers. We employ similar approaches to elucidating how CRISPR/as9-mediated gene editing is accomplished using single-stranded DNA templates. A third area of concern is to understand how the DNA damage checkpoint is regulated. We wish to determine how the DNA damage response affects DSB repair and how the DNA damage checkpoint is maintained and turned off. These studies will provide new insights and guidance in defining the DSB repair and checkpoint signaling in human cells. Finally, we will continue our investigation of the mechanisms by which Borrelia initiates and mediates gene conversion events between one expressed gene and a set of adjacent pseudogenes.