DNA replication is essential to maintain the genome of all organisms. During each round of cell division, eukaryotic cells must establish hundreds to thousands of replication forks that coordinately replicate each chromosome. The events that assemble the multi-enzyme replisomes that act at each replication fork are tightly regulated to ensure that each chromosome is replicated exactly once. Consistent with their importance, defects in or misregulation of replication initiation proteins are known to lead to cancer and developmental abnormalities. It is critical to understand how the essential event of replication initiation occurs and how this event leads to the appropriate assembly of the replication machinery. In recent years, significant advances have been made in our understanding of the initial event of this process, the selection of the sites of replication initiation (origins of replication) through the loading of the replicative DNA helicase. In contrast, we know significantly less about the events that activate these loaded helicases and the subsequent assembly of the DNA replication machinery. The proposed research will exploit a novel in vitro assay that recapitulates the events of DNA replication initiation from a defined origin of replication. We will use this assay and new assays derived from it to address fundamental questions concerning the initiation of replication. We will primarily focus on the committed step of replication initiation, DNA helicase activation.
In specific aim one, we will develop assays to address how the loaded helicase is primed for helicase activation but maintained in an inactive state. In the second aim, we will use novel DNA substrates and assays to determine how the numerous proteins required for helicase activation contribute to this event, with a focus on the known helicase-activating protein, Cdc45. In the last aim, we will address how helicase activation and DNA unwinding are coordinated with the initiation of DNA synthesis to prevent the generation of excess single-stranded DNA and activation of DNA-damage checkpoint.

Public Health Relevance

Project Narrative: The accurate and complete replication of chromosomal DNA is essential to maintain cell identity and prevent genome changes that can lead to cancer or developmental defects. We have developed novel assays that recapitulate these events in a test tube which we will exploit to determine how the molecular machines that replicate chromosomal DNA are correctly assembled.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Janes, Daniel E
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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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
Kang, Sukhyun; Warner, Megan D; Bell, Stephen P (2014) Multiple functions for Mcm2-7 ATPase motifs during replication initiation. Mol Cell 55:655-65
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
Takara, Thomas J; Bell, Stephen P (2011) Multiple Cdt1 molecules act at each origin to load replication-competent Mcm2-7 helicases. EMBO J 30:4885-96
Tsakraklides, Vasiliki; Bell, Stephen P (2010) Dynamics of pre-replicative complex assembly. J Biol Chem 285:9437-43
Francis, Laura I; Randell, John C W; Takara, Thomas J et al. (2009) Incorporation into the prereplicative complex activates the Mcm2-7 helicase for Cdc7-Dbf4 phosphorylation. Genes Dev 23:643-54
Chen, Shuyan; de Vries, Milan A; Bell, Stephen P (2007) Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2-7 loading. Genes Dev 21:2897-907
Bell, Oliver; Wirbelauer, Christiane; Hild, Marc et al. (2007) Localized H3K36 methylation states define histone H4K16 acetylation during transcriptional elongation in Drosophila. EMBO J 26:4974-84

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