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Regulated eukaryotic DNA replication origin firing with purified proteins

Author

Listed:
  • Joseph T. P. Yeeles

    (Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK)

  • Tom D. Deegan

    (Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK)

  • Agnieszka Janska

    (Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK)

  • Anne Early

    (Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK)

  • John F. X. Diffley

    (Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK)

Abstract

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45–MCM–GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Suggested Citation

  • Joseph T. P. Yeeles & Tom D. Deegan & Agnieszka Janska & Anne Early & John F. X. Diffley, 2015. "Regulated eukaryotic DNA replication origin firing with purified proteins," Nature, Nature, vol. 519(7544), pages 431-435, March.
    • Handle: RePEc:nat:nature:v:519:y:2015:i:7544:d:10.1038_nature14285
    DOI: 10.1038/nature14285
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    Citations

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    Cited by:

    1. Aftab Amin & Cheung Man Hei & Chun Liang & Aftab Amin & Cheung Man Hei & Chun Liang & Chun Liang & Aftab Amin & Cheung Man Hei & Chun Liang, 2019. "DNA Replication-Initiation Proteins in Eukaryotic Cells," Biomedical Journal of Scientific & Technical Research, Biomedical Research Network+, LLC, vol. 22(5), pages 17042-17049, December.
    2. Dayana E. Salas-Leiva & Eelco C. Tromer & Bruce A. Curtis & Jon Jerlström-Hultqvist & Martin Kolisko & Zhenzhen Yi & Joan S. Salas-Leiva & Lucie Gallot-Lavallée & Shelby K. Williams & Geert J. P. L. K, 2021. "Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
    3. Jiaxuan Cheng & Ningning Li & Yunjing Huo & Shangyu Dang & Bik-Kwoon Tye & Ning Gao & Yuanliang Zhai, 2022. "Structural Insight into the MCM double hexamer activation by Dbf4-Cdc7 kinase," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    4. Humberto Sánchez & Zhaowei Liu & Edo Veen & Theo Laar & John F. X. Diffley & Nynke H. Dekker, 2023. "A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    5. Zaida Vergara & María S. Gomez & Bénédicte Desvoyes & Joana Sequeira-Mendes & Kinda Masoud & Celina Costas & Sandra Noir & Elena Caro & Victoria Mora-Gil & Pascal Genschik & Crisanto Gutierrez, 2023. "Distinct roles of Arabidopsis ORC1 proteins in DNA replication and heterochromatic H3K27me1 deposition," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    6. Sai Li & Michael R. Wasserman & Olga Yurieva & Lu Bai & Michael E. O’Donnell & Shixin Liu, 2022. "Nucleosome-directed replication origin licensing independent of a consensus DNA sequence," Nature Communications, Nature, vol. 13(1), pages 1-15, December.

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