Studies over the past decade have revealed that the homologous recombination process of Break-Induced Replication (BIR) is fundamentally important in re-starting stalled or broken replication forks as well as in maintaining eukaryotic telomeres in the absence of telomerase. In addition, BIR appears to be important in the creation of chromosome rearrangements between homologous sequences, including deletions and nonreciprocal translocations. In the model organism, Saccharomyces cerevisiae, there are two Rad52- dependent, recombination-dependent DNA replication processes, one of which requires the Rad51 recombinase and a second that utilizes the Mre11-Rad50-Xrs2 complex and Rad59. This proposal focuses on the more efficient, Rad51-dependent BIR pathway. Experiments are proposed to understand the detailed molecular mechanisms and genetic requirements to assemble a repair replication fork that can copy a template chromosome for at least several hundred kb. A haploid strain in which BIR produces a nonreciprocal translocation will be used to monitor in real time the repair of a site-specific double-strand break, induced by a meganuclease such as HO or l-Scel. The DNA sequence homology requirements for BIR will be determined and possible sequence-specific barriers to BIR progression will be analyzed. Both intermediates in the DNA and the recruitment of proteins to the site of DSB repair can be followed in real time. The role of proteins important in establishing and elongating normal DNA replication will be evaluated for their role in the establishment and progression of the repair replication fork, which can be analyzed in post-start G1 cells or in G2 cells, when there is no competing normal replication. The repair replication fork will be analyzed to learn about its processivity and fidelity. Isotope density transfer experiments will be used to determine if the newly replicated DNA strands are displaced from their template or remain as semi- conservative replication products. Finally, systematic genome screens will be used to identify new genes that play key roles in BIR and in telomere maintenance in the absence of telomerase. ? ? ?
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