Friday, April 26, 2024
4/26/2024
Project Summary The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans, and is a fundamental step required for genomic stability in all cells. Yet until recently, the question of how this process occurs had not been characterized, in any cell type. We recently demonstrated that the completion of DNA replication in E. coli involves an enzymatic system that effectively limits cellular replication when it reaches its `doubling point' by allowing converging replication forks to transiently pass each other before the excess, over- replicated regions are incised, resected, and joined. The completion reaction requires RecBCD and involves several proteins associated with repairing double-strand breaks including, SbcC- SbcD-ExoI. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. Many of bacterial proteins required to complete DNA replication have clear homologs in eukaryotes. Bacterial SbcC-SbcD and ExoI are highly conserved with Mre11-Rad50 and CtIP, a poorly characterized nuclease complex essential for genome stability, normal development, and viability in mammals. Here, we propose to extend these important findings to eukaryotic cells, and to determine the enzymatic pathway that catalyzes the completion of replication in the model eukaryote, Saccharomyces cerevisiae. We employ a novel approach that enables us to identify, map, and characterize sites where replication completes directly on eukaryotic chromosomes. We will use this approach to establish the essential role of Mre11-Rad50-CtIP during cellular replication, determine the enzymes required for the eukaryotic completion reaction, and identify synthetic lethal genes in completion mutants, that can be targeted for potential therapeutics. The results of these studies will identify a fundamental, yet heretofore unstudied, aspect of cellular replication that is central to genome stability.
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