The timely complete and accurate replication of the genome is essential to the normal proliferation of all eukaryotic cells. Either too much or too little replication can have lethal consequences for the cell. Accordingly, the initiation of DNA replication is tightly coordinated with progression through the cell division cycle. The overall goal of this proposal is to define the molecular mechanisms that direct the initiation of eukaryotic DNA replication and determine how they are integrated into and regulated by the cell cycle machinery. To ensure that replication does not initiate more than once per cell cycle, eukaryotic cells divide the replication initiation into two temporally distinct events: origin selection and origin activation. During G1-phase, all potential origins of replication are selected through the assembly of pre-replication complexes (pre-RCs). During S-phase, these sites are activated to initiate replication by the formation of two higher order complexes: the pre-initiation complex (pre-IC) and the replisome. During pre-RC formation the six-protein, replicative DNA helicase (the Mcm2-7 complex) is loaded onto origin DNA in an inactive state. Formation of the pre-IC activates the Mcm2-7 helicase and the helicase-dependent unwinding of origin DNA that is required for replisome assembly. Using biochemical assays that recapitulate pre-RC formation and mutant and modified proteins involved in these events Dr. Bell will: 1. Determine the mechanism of Mcm2-7 origin loading. 2. Determine the impact of nucleosomes on pre-RC formation. 3. Reconstitute and dissect pre-IC formation. These studies will be performed using the yeast S. cerevisiae rather than human cells. The availability of highly defined sequences that act as origins of DNA replication and powerful cell cycle tools make studies of DNA replication in this organism particularly advantageous. Nevertheless, the highly conserved nature of eukaryotic DNA replication indicates that progress made in this yeast will be translated rapidly to human cells. New understanding of the events of replication initiation will lead to candidate targets for anti-fungal compounds. Moreover, the understanding eukaryotic replication provided by these yeast studies will direct studies to identify inhibitors of the same events in humans, which represent important candidates for chemotherapeutic agents. Project Narrative In this proposal Dr. Bell will study the fundamental events that control the duplication of animal cell chromosomes. In particular, he will investigate how the hundreds, if not thousands, of sites at which this process is initiated are selected and activated to start chromosome duplication at the appropriate times. The findings will provide fundamentally important information that will be relevant to the proliferation of all animal cells including humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052339-16
Application #
7846908
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Hagan, Ann A
Project Start
1995-05-01
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
16
Fiscal Year
2010
Total Cost
$283,204
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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
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
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
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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