The timely and accurate replication of the genome is essential to the normal proliferation of all eukaryotic cells. Accordingly the initiation of DNA replication is carefully coordinated with the progress of the cell cycle. The long-term objective of this proposal is to determine the events that select the sites of DNA replication and activate the initiation process. Control of DNA replicaiton is tightly linked to the formation and activation of the pre-replicative complex (pre-RC) that includes the origin recognition complex (ORC), Cdc6 and the Mcm2-7 complex (the putative replicative DNA helicase). Using a combination of new in vitro assays for pre- RC formation and mutants in ORC, Cdc6 and the Mcm2-7 proteins Dr. Bell will: 1. Determine the components of the pre-RC and the mechanism of pre-RC formation. 2. Determine the how ATP controls the formation of the pre-RC. 3. Determine how cyclin dependent kinases and ORC work together to prevent origins from re-initiating at inappropriate times during the cell cycle. 4. Develop new in vitro assays for the events leading to replication initiation from a defined origin of replication. These studies will be performed using the yeast S. cerevisiae, however, the highly conserved nature of eukaryotic DNA replication indicates progress in yeast will rapidly translated to an understanding of the same processes in human cells. The rapid progress that can be made in S. cerevisiae make it an ideal organism to perform these studies. New understanding of these proteins will lead to strong candidate targets for antifungal compounds and potential markers to identify cancer cells. In addition, the understanding of the yeast replication proteins gained in these studies will direct studies to identify inhibitors of the human analogs of these proteins, which represent important targets for novel chemotherapeutic compounds. ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-SSS-U (04))
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Dearolf, Charles R
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Bell, Stephen P (2017) Rethinking origin licensing. Elife 6:
Ticau, Simina; Friedman, Larry J; Champasa, Kanokwan et al. (2017) Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing. Nat Struct Mol Biol 24:309-315
Bell, Stephen P; Labib, Karim (2016) Chromosome Duplication in Saccharomyces cerevisiae. Genetics 203:1027-67
Duzdevich, Daniel; Warner, Megan D; Ticau, Simina et al. (2015) The dynamics of eukaryotic replication initiation: origin specificity, licensing, and firing at the single-molecule level. Mol Cell 58:483-94
Ticau, Simina; Friedman, Larry J; Ivica, Nikola A et al. (2015) Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. Cell 161:513-525
Kang, Sukhyun; Warner, Megan D; Bell, Stephen P (2014) Multiple functions for Mcm2-7 ATPase motifs during replication initiation. Mol Cell 55:655-65
Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B et al. (2014) A conserved MCM single-stranded DNA binding element is essential for replication initiation. Elife 3:e01993
Bell, Stephen P; Kaguni, Jon M (2013) Helicase loading at chromosomal origins of replication. Cold Spring Harb Perspect Biol 5:
Heller, Ryan C; Kang, Sukhyun; Lam, Wendy M et al. (2011) Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. Cell 146:80-91
Chen, Shuyan; Bell, Stephen P (2011) CDK prevents Mcm2-7 helicase loading by inhibiting Cdt1 interaction with Orc6. Genes Dev 25:363-72

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