A long-term goal of my research program is to understand the molecular mechanisms underlying the initiation of cellular DNA replication. Initiation is a defining commitment to cell proliferation;inappropriate onset of replication can lead to changes in gene copy number, DNA damage, and genetic instabilities. From a biomedical perspective, initiation is a keystone pathway that should be susceptible to therapeutic intervention for controlling bacterial infections and cancers;however, our understanding of initiation processes is insufficiently complete to advance such efforts. The present application focuses on the initiation of DNA replication in bacteria. Although a basic framework for this process has been in place for more than 25 years, its mechanistic principles have remained highly enigmatic. By employing an innovative mix of structural methods, new biochemical assays, and analytic technologies, we will answer fundamental questions involving how initiation proteins collaborate to open a bubble in a replication origin and deposit ring-shaped helicases onto the DNA. We will determine in molecular detail: 1) how the DnaA initiator melts DNA and whether nascent single DNA strands subsequently regulate DnaA activity, 2) whether the hexameric DnaB helicase autoregulates its DNA unwinding activity, and 3) how the DnaC helicase loader, together with DnaA and the DnaG primase, systematically cooperate in shepherding the helicase onto DNA. The outcome of the proposed studies will be a stepwise structural and functional picture of the major steps involved in converting a duplex chromosomal region into a bidirectional replication fork. These findings in turn will: 1) define new principles for both the field of DNA replication and the broadr action of ATP-dependent machines and switches, and 2) establish new assays and models for advancing drug-discovery efforts that target initiation systems.

Public Health Relevance

The initiation of DNA replication is central to cell proliferation and survival. Initiaton pathways should be susceptible to therapeutic intervention against bacterial infections and cancers;however, limitations in our understanding of basic initiation mechanisms impede this goal. The proposed work will provide key molecular models and assays for accelerating antibacterial-discovery efforts that target initiation proteins and activities.

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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Preusch, Peter C
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Johns Hopkins University
Schools of Medicine
United States
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Costa, Alessandro; Renault, Ludovic; Swuec, Paolo et al. (2014) DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome. Elife 3:e03273
O'Shea, Valerie L; Berger, James M (2014) Loading strategies of ring-shaped nucleic acid translocases and helicases. Curr Opin Struct Biol 25:16-24
Strycharska, Melania S; Arias-Palomo, Ernesto; Lyubimov, Artem Y et al. (2013) Nucleotide and partner-protein control of bacterial replicative helicase structure and function. Mol Cell 52:844-54
Duderstadt, Karl E; Berger, James M (2013) A structural framework for replication origin opening by AAA+ initiation factors. Curr Opin Struct Biol 23:144-53
Arias-Palomo, Ernesto; O'Shea, Valerie L; Hood, Iris V et al. (2013) The bacterial DnaC helicase loader is a DnaB ring breaker. Cell 153:438-48
Costa, Alessandro; Hood, Iris V; Berger, James M (2013) Mechanisms for initiating cellular DNA replication. Annu Rev Biochem 82:25-54
Costa, Alessandro; Ilves, Ivar; Tamberg, Nele et al. (2011) The structural basis for MCM2-7 helicase activation by GINS and Cdc45. Nat Struct Mol Biol 18:471-7
Duderstadt, Karl E; Chuang, Kevin; Berger, James M (2011) DNA stretching by bacterial initiators promotes replication origin opening. Nature 478:209-13
Lyubimov, Artem Y; Strycharska, Melania; Berger, James M (2011) The nuts and bolts of ring-translocase structure and mechanism. Curr Opin Struct Biol 21:240-8
Duderstadt, Karl E; Mott, Melissa L; Crisona, Nancy J et al. (2010) Origin remodeling and opening in bacteria rely on distinct assembly states of the DnaA initiator. J Biol Chem 285:28229-39

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