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Structure and Function Studies of Replication Initiation Factors

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The Initiation of DNA Replication in Eukaryotes

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Abstract

We have used negative stain EM and cryo-EM to visualize step by step the replication initiation events in S. cerevisiae, as the process is driven forward by the interplay of a dozen or so macromolecular initiation factors, leading to the establishment of pre-replication complexes (pre-RC) at each origin of DNA replication. This work took advantage of our ability to reconstitute the Mcm2-7 loading reaction with purified proteins. We determined the architecture of several previously known replication initiation complexes such as ORC, ORC-Cdc6 on DNA, and the Mcm2-7 double hexamer. We also captured by EM reaction intermediates such as the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) and the ORC-Cdc6-Mcm2-7-Mcm2-7 (OCMM) that had evaded previous biochemical identification. In this chapter, we describe what we have learnt about the structure and interaction with origin DNA of the replication initiators. We further discuss what may be expected in the coming years as cryo-EM is becoming a near-atomic resolution structural tool, thanks to the recent advent of the direct electron detector.

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Acknowledgements

Many people in the labs of Huilin Li, Christian Speck, and Bruce Stillman have helped in this work. The work was supported by National Institutes of Health grant numbers GM45436 (to B.S.) and GM74985 (to H.L.) and the United Kingdom Biotechnology and Biological Sciences Research Council (to C.S.).

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Authors and Affiliations

  1. Biosciences Department, Brookhaven National Laboratory, Upton, NY, 11973, USA

    Jingchuan Sun, Zuanning Yuan & Huilin Li

  2. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, 11794, USA

    Zuanning Yuan & Huilin Li

  3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724, USA

    Bruce Stillman

  4. DNA Replication Group, Imperial College London, Faculty of Medicine, Institute of Clinical Sciences, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK

    Christian Speck

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  1. Jingchuan Sun
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  2. Zuanning Yuan
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  3. Bruce Stillman
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  4. Christian Speck
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  5. Huilin Li
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Correspondence to Bruce Stillman , Christian Speck or Huilin Li .

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  1. Florida State Univ. College of Medi, TALLAHASSEE, Florida, USA

    Daniel L. Kaplan

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Sun, J., Yuan, Z., Stillman, B., Speck, C., Li, H. (2016). Structure and Function Studies of Replication Initiation Factors. In: Kaplan, D. (eds) The Initiation of DNA Replication in Eukaryotes. Springer, Cham. https://doi.org/10.1007/978-3-319-24696-3_21

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