Analysis of Break-Induced Replication

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DESCRIPTION (provided by applicant): Summary The repair of broken chromosomes is essential for maintenance of chromosome structure and genome stability. This proposal focuses on delineating the mechanisms of repair of double-strand chromosome breaks (DSBs) by break-induced replication (BIR). BIR plays a key role in restarting stalled and broken DNA replication forks, in maintaining telomeres in the absence of the telomerase enzyme and in the newly discovered phenomenon of single chromosome shattering, known as chromothripsis, which is associated with human cancers and developmental diseases. A microhomology-mediated BIR (MM-BIR) process has been hypothesized to account for long-distance template switching events that lead to the joining of distant sequences to create novel gene fusions. Previous work from this laboratory has identified roles for replication proteins Pol32 and PCNA that are essential for BIR but not for normal replication or other types of DSB repair in the model organism, budding yeast. The proposed project to continue this study will focus on two main topics. First, the molecular mechanism of BIR will be pursued, creating site-specific DSBs by an inducible endonuclease. A novel assay has been developed to study surprisingly frequent template jumps during BIR both between homologous sequences in distant locations or between homeologous sequences (highly mismatched sequences that permit study of MM-BIR). This assay permits one to distinguish between the way in which a broken DNA end locates and recombines with a distant sequence by strand invasion and how the subsequent replicating strands in BIR then can jump to a third location. The role of mismatch repair proteins in discouraging BIR between diverged sequences will be investigated. Evidence that template jumping uses a different mechanism from the initial strand invasion step will be pursued. The possibility that the jump does not require the Rad51 recombinase protein will be explored. Special attention will be devoted to the role of Rdh54, the first protein that is specifically required for template jumps but competent for simple BIR and other recombination events. Whether repair of a broken replication fork, created by nicking a specific strand with a CRISPR endonuclease, obeys the same rules as the ectopic model systems and telomere-repair events that have so far been studied is a fundamentally important question. A second major goal will be to examine translocations and rearrangements in which there is little or no homology at the repair junctions. A novel assay involving creation o a functional intron by joining distant sequences will be used. Finally, template jumps into unrelated sequences will be recovered and analyzed by DNA sequencing in order to better define how much homology and adjacent homeology is required for microhomology-mediated events. These studies are highly significant in understanding the origins of chromosome rearrangements associated with human disease, including the loss of heterozygosity caused by the formation of nonreciprocal translocations, segmental duplications and the astonishing rearrangements in chromosomes experiencing chromothripsis.