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 from 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 and human cell proteins, 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. Biochemical and structural studies will elucidate how molecular machines that are driven by ATP function to load ring shaped protein onto double stranded DNA and how origin recognition has evolved in eukaryotes.

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 at origin of DNA replication throughout the genome that renders each origin competent for subsequent initiation of DNA replication, including assembly of a pre-Initiation Complex. This project will examine how the initiation of DNA replication occurs and how it is regulated, including studies at the structural level.

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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Reddy, Michael K
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Cold Spring Harbor Laboratory
Cold Spring Harbor
United States
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On, Kin Fan; Jaremko, Matt; Stillman, Bruce et al. (2018) A structural view of the initiators for chromosome replication. Curr Opin Struct Biol 53:131-139
Yuan, Zuanning; Riera, Alberto; Bai, Lin et al. (2017) Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1. Nat Struct Mol Biol 24:316-324
Tocilj, Ante; On, Kin Fan; Yuan, Zuanning et al. (2017) Structure of the active form of human origin recognition complex and its ATPase motor module. Elife 6:
Noguchi, Yasunori; Yuan, Zuanning; Bai, Lin et al. (2017) Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model. Proc Natl Acad Sci U S A 114:E9529-E9538
Sheu, Yi-Jun; Kinney, Justin B; Stillman, Bruce (2016) Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression. Genome Res 26:315-30
Stillman, Bruce (2015) Reconsidering DNA Polymerases at the Replication Fork in Eukaryotes. Mol Cell 59:139-41
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
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
Rossmann, Marlies P; Stillman, Bruce (2013) Immunoblotting histones from yeast whole-cell protein extracts. Cold Spring Harb Protoc 2013:625-30

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