Genetic instability plays a critical role in carcinogenesis, making knowledge about the mechanisms that lead to genome rearrangements and mutagenesis a critical tool in the fight against cancer. This project is focused on a novel type of DNA synthesis, migrating-bubble DNA synthesis (MiBS), which promotes bursts of genomic instability, including hyper-mutagenesis, translocations, and copy number variations. In stark contrast to S- phase replication, MiBS is initiated at a double-strand break (DSB) site rather than at a replication origin, is carried out by a migrating bubble rather than by a replication fork, and leads to conservative inheritance of newly synthesized DNA. This proposal aims to unravel the molecular mechanism of MiBS and to determine how MiBS promotes various types of genetic instabilities characteristic of human cancers. To study MiBS, we will use a dependable and powerful system in yeast, Saccharomyces cerevisiae, where a single DSB initiated by a site-specific HO endonuclease is repaired by break-induced replication (BIR), an important DSB repair pathway which proceeds through MiBS. More specifically, a DSB is repaired by invasion of one free end of broken DNA into the homologous chromosome followed by DNA synthesis mediated by MiBS that proceeds for approximately 100 kilobases to the end of the homologue, resulting in a repaired molecule with a normal telomere. We will use direct physical methods, including two-dimensional gel electrophoresis, dynamic molecular combing, and electron microscopy to determine the mechanism of MiBS and to characterize the roles of replication proteins that are responsible for it. We will further determine the mechanism of increased mutagenesis promoted by MiBS, employ sensitive genetic analyses to fully characterize the role of DNA polymerases in MiBS-associated hypermutability, and assess the role of MiBS in the formation of mutation clusters using whole-genome DNA sequencing. Importantly, the results of these investigations will shed light on a mechanism of regional hyper-mutability, kataegis, which has recently been described in various types of cancer. Finally, we will determine the role of MiBS in promoting complex GCRs similar to those associated with chromothripsis, a cancer-related phenomenon that involves massive genomic changes localized to a single chromosome. Preliminary results obtained in the PI's lab suggest that chromothripsis-like GCRs may occur when DSB repair switches from MiBS to microhomology-mediated BIR (MMBIR). The proposed research will unravel the mechanism mediating switches from MiBS to MMBIR, including the role of translesion DNA polymerases in this process, and will determine the role of MMBIR in formation of GCRs. Overall, the results of this proposed research are expected to establish a novel concept: the notion that a burst of genetic instabilities that can lead to cancer may result from an unusual type of replication (MiBS) rather than from a continuing accumulation of small genetic changes during semi-conservative S-phase replication.