The goal of this research is to determine the mechanism and regulation of the initiation of DNA replication in eukaryotic cells. It is clear that for maintenance of the integrity of the genome frm one cell generation to the next, DNA and its associated chromatin structures must be duplicated in a highly controlled and accurate manner. Interruption of these controls may promote genome instability and lead to neoplastic transformation in somatic cells or result in mutations in the germ line that can cause many different disorders. Moreover, the DNA replication proteins represent tangible targets for therapeutic intervention and diagnosis of proliferation of cancer cells, and other proliferative disorders. The initiator protein (ORC) cooperates with a series of DNA replication proteins, including Cdc6, Cdt1 and the MCM2-7 hexamer to establish at origins of DNA replication a pre-Replicative Complex (pre-RC) that facilitates later initiation of DNA synthesis at each origin. Recent progress has enabled the assembly of the pre-RC in vitro with purified proteins. The proposed research in this application will investigate, using the yeast S. cerevisiae, how the initiation of DNA replication occurs following pre-RC assembly and how this process is regulated by the Cdc7-Dbf4 (DDK) protein kinase and by an intrinsic inhibitor of initiation of DNA replication within the Mcm4 subunit of the MCM2-7 complex. The proposed research will also investigate how the core histones within nucleosomes, the fundamental structural unit of chromatin in eukaryotic cells, are disrupted during DNA replication and transferred to the leading and lagging strands of the newly synthesized DNA.

Public Health Relevance

DNA replication is the process whereby the genome is duplicated prior to segregation of the resulting sister chromatids during mitosis or meiosis. During mitotic exit or during G1 phase, a pre-Replicative Complex is assembled each origin of DNA replication that renders each origin competent for subsequent initiation of DNA replication. This project will examine how the initiation of DNA synthesis occurs and how it is regulated, as well as investigate how chromatin is inherited.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045436-22
Application #
8470651
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
1991-07-01
Project End
2016-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
22
Fiscal Year
2013
Total Cost
$676,199
Indirect Cost
$318,422
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724
Sun, Jingchuan; Fernandez-Cid, Alejandra; Riera, Alberto et al. (2014) Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function. Genes Dev 28:2291-303
Sheu, Yi-Jun; Kinney, Justin B; Lengronne, Armelle et al. (2014) Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression. Proc Natl Acad Sci U S A 111:E1899-908
O'Donnell, Michael; Langston, Lance; Stillman, Bruce (2013) Principles and concepts of DNA replication in bacteria, archaea, and eukarya. Cold Spring Harb Perspect Biol 5:
Sun, Jingchuan; Evrin, Cecile; Samel, Stefan A et al. (2013) Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Nat Struct Mol Biol 20:944-51
Sun, Jingchuan; Kawakami, Hironori; Zech, Juergen et al. (2012) Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy. Structure 20:534-44
Rossmann, Marlies P; Luo, Weijun; Tsaponina, Olga et al. (2011) A common telomeric gene silencing assay is affected by nucleotide metabolism. Mol Cell 42:127-36
Sheu, Yi-Jun; Stillman, Bruce (2010) The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Nature 463:113-7
Yu, Shirong; Smirnova, Julia B; Friedberg, Errol C et al. (2009) ABF1-binding sites promote efficient global genome nucleotide excision repair. J Biol Chem 284:966-73
Li, Qing; Fazly, A M; Zhou, Hui et al. (2009) The elongator complex interacts with PCNA and modulates transcriptional silencing and sensitivity to DNA damage agents. PLoS Genet 5:e1000684
Evrin, Cecile; Clarke, Pippa; Zech, Juergen et al. (2009) A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proc Natl Acad Sci U S A 106:20240-5

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