Project Summary / Abstract Mutagenesis resulting from replication failure is a direct cause of tissue dysfunction and cancer when it occurs in somatic tissues and of de novo and inherited genetic diseases when it occurs in gametogenic stem cells or meiosis. A primary mutagenic outcome of replication failure is structural variant (SV) formation, especially copy number variants (CNVs), which create large changes in genomic content in single mutational steps. Fundamental gaps in knowledge exist regarding the DNA repair mechanisms that lead to SV formation. While multiple mechanisms may be involved, models that derive from template switching, break-induced replication (BIR), and other forms of double-strand break (DSB) repair have been forwarded to account for a large proportion of human CNVs but lack direct experimental evidence. Our prior work has shown that incomplete replication leads to a high frequency of CNVs in human cells, with hotspots in large, transcribed genes corresponding to common fragile sites (CFSs) that provide a model system for characterizing SV formation mechanisms. Recent literature has revealed much about the damage response pathways that promote proper completion of replication. One finding was Mitotic DNA Synthesis (MiDAS), where failed replication in S is rescued by a conservative form of replication activated as late as mitosis. As a BIR-like pathway, MiDAS accuracy is thought to be low such that the temporal association between MiDAS and CFS expression suggests a potential mechanistic link to SV formation. Our major goals are to explore the relationships between replication rescue, end-joining, BIR, other forms of DSB repair, and SV formation, how CFS expression relates to CNV formation, and how extensible observations at CFS/CNV hotspot loci are to SV formation genome wide. Our central hypothesis is that hotspot CNV formation occurs during replication rescue, either via MiDAS or an alternative pathway to MiDAS, notably, theta-mediated end joining (TMEJ). A driving rationale is that we must monitor SV formation in real time as a primary experimental outcome, something we have been uniquely dedicated to doing. Our approach will therefore apply our recent technology advances for directly detecting rare SV junctions in experimental samples to provide answers to longstanding questions about the origins of human SVs. We will address our goals through three specific aims to (1) Identify the precise cell cycle stage(s) when structural variants form following replication stress; (2) Establish the replication rescue and DNA repair pathways that create structural variant junctions; and (3) Extend mitotic SV formation mechanisms from CFSs to the whole genome and BRCA2 deficiency. The combination is significant as it will provide direct experimental tests of the mechanisms that execute SV formation in at-risk genomic loci and extend those findings to multiple genomic regions and cell lineages relevant to both somatic and heritable ...