Candida glabrata is an opportunistic fungal pathogen associated with high mortality and whose incidence is increasing due to its high frequency of resistance to the widely used azole antifungal class. C. glabrata also rapidly evolves resistance to echinocandins and can become multi-drug resistant and thus virtually impossible to treat. Drug resistance in C. glabrata is acquired via specific genetic variants. C. glabrata is also notable for its remarkable genetic diversity, manifested by a variety of karyotypes and high levels of short nucleotide polymorphisms (SNPs) among strains. However, how C. glabrata facilitates genetic instability is almost entirely unknown. A major source of genetic instability in all examined biological systems are DNA double-strand breaks (DSBs), which mediate chromosome rearrangements and are associated with high rates of point mutations in nearby regions. Thus, both chromosome rearrangements and SNP variation across C. glabrata strains are consistent with DNA DSBs being the major source of this genetic diversity. Indeed, our preliminary studies showed that C. glabrata experiences DNA breaks and develops chromosome rearrangements and drug-resistant mutations during its interaction with host cells, e.g., while residing in macrophages, and that deletion of DSB repair gene RAD51 in C. glabrata significantly increases the emergence of drug-resistant mutants in the mouse gastrointestinal colonization model. This proposal is based on the hypothesis that C. glabrata has evolved mechanisms that facilitate genetic instability upon DNA damage and that to understand these mechanisms it is necessary to understand how C. glabrata generates and processes DNA DSBs. In Specific Aim 1, we propose to use DSB chromatin immunoprecipitation followed by next generation sequencing (DSB-ChIP-seq) and END- seq (a highly sensitive, unbiased next-generation sequencing technique for quantitatively mapping DSBs at nucleotide resolution across the genome) to identify “fragile” loci prone to DSB formation in C. glabrata, based on the hypothesis that these loci are the most likely mediators of genetic instability. In Specific Aim 2, we will use DSB-ChIP followed by mass spectrometry (DSB-ChIP-MS) to identify C. glabrata proteins that mediate DSB transactions. In both Aims, the roles of selected identified loci/genes in DSB formation/processing and genome stability will be validated experimentally. The proposed study will fill a large gap in knowledge and provide information essential for understanding how C. glabrata promotes genetic diversity and evolves drug-resistant variants. Karyotype instability and aberrant DSB repair are also hallmarks of many cancers, and in that context understanding the mechanisms underlying DSB formation and processing has been instrumental in developing effective therapeutic approaches targeting DSB repair mechanisms, e.g., by using PARP inhibitors. Thus, this proposal will provide the first understanding of DSB formation...