DNA replication restart pathways reload cellular DNA replication complexes onto replication forks that have been prematurely abandoned. These pathways form an essential link between DNA repair (often recombinational repair) and replication. The proteins that drive these reactions, referred to as the primosome or the Replication Restart Proteins, must recognize the structures of abandoned replication forks and reload the DNA replication machinery specifically at these sites. This process is heavily regulated to ensure loading fidelity and to avoid over-replication that could arise from initiating replication at improper DNA structures. In spite of the broad biological importance of this process, the mechanisms underlying DNA replication restart and its regulation remain poorly understood. Additionally, the mechanisms by which replication restart pathways are integrated with core cellular DNA repair processes are currently unknown. Our proposal combines structural, biochemical, and genetic approaches to define the mechanisms of DNA replication restart in complementary ways. Our first overall objective of this application is to determine the structural mechanisms that govern DNA replication restart, from recognition of abandoned DNA replication forks, to primosome assembly, and finally to reloading the first component of the DNA replication machinery.
Aim 1 takes advantage of our preliminary data to define structural snapshots of each step in DNA replication restart. Our second goal is to systematically define the genetic mechanisms that support DNA replication restart.
Aim 2 will identify the genes that coordinate double-strand DNA break repair with replication restart and will define circumstances under which the major replication restart pathways are utilized in cells. Additionally, the mechanisms underlying replication restart suppressors will be examined.
DNA replication restart is an important process to study from a human health perspective because knowing more about DNA replication will help to identify targets to kill selectively the deleterious, malignant or disease causing cells that may plague our bodies (e.g., tumors, cancers, pathogenic organisms). Conversely, this knowledge may also help to augment basic health and longevity of our bodies by the design of better antibiotics. This may be translated into a longer, healthier life for individuals. Key words: primosome, replication restart, structural biology, biochemistry, genetics.
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