Accurate repair of DNA lesions is paramount to the survival of cells and to maintain their genomic stability. Double-strand DNA breaks (DSBs) are the most lethal DNA lesion, and cells have evolved a variety of mechanisms for their repair. While some DSB repair pathways are accurate, others can destabilize the genome by creating mutations or chromosome rearrangements associated with cancer and other human diseases. Our long-term goal is to identify factors that drive DSB repair into the maelstrom of deleterious DNA repair pathways, and to characterize their molecular mechanisms. We focus on two such high-risk DSB repair pathways: 1) break-induced replication (BIR), an unusual type of long-tract repair DNA synthesis that promotes bursts of genetic instabilities; and 2) microhomology-mediated BIR (MMBIR), a replicative pathway involving multiple template-switching events at positions of microhomologies that yields complex genomic rearrangements. We will use an extensively characterized, powerful yeast system to study repair of a site- specific HO-endonuclease-induced DSB to inform the design of studies in other systems. MIRA support enabled significant progress in our characterization of BIR and MMBIR, including development of several innovative tools. One of them, which we named AMBER (Assay for Monitoring BIR Elongation Rate), is a droplet-digital-PCR-based method to measure BIR kinetics with unprecedented resolution. Using AMBER during the next MIRA support cycle will allow us to identify the specific steps of BIR that are controlled by our newly identified BIR driver protein candidates, including spindle assembly checkpoint proteins. We will also use AMBER in our sensitive yeast BIR system to unravel the mechanisms of BIR regulation following its collision with various replication obstacles, including characterizing the role of Rad52-dependent single-strand annealing for BIR re-start after collision. The obtained results will shed light on the mechanism of Rad51- independent BIR in yeast, which is a pathway that is likely similar to BIR events described in mammals. Another approach that we developed with MIRA support enabled the detection of BIR events based on long mutation clusters formed by BIR occurring in the presence of APOBEC (cytidine deaminase), and we propose to apply this methodology here to detect BIR during yeast meiosis. Determining how frequently mutagenic BIR might be used to repair meiotic DSBs is important because similar events can lead to birth defects in humans. Finally, our new software, MMBSearch—developed based on our characterization of MMBIR in yeast—will be used to identify specific conditions that predispose human cells to MMBIR events, which we recently found to be frequent in cancer, but rare in non-cancerous cells. Applying MMBSearch to whole-genome sequencing data will identify specific cancers, cell types, chromosomal locations and environmental stressors that promote MMBIR. Overall, this research program will pr...