CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol ɛ, and GINS in budding yeast

  1. Hiroyuki Araki1,2,6
  1. 1Division of Microbial Genetics, National Institute of Genetics, Research Organization of Information and Systems, ROIS, Mishima 411-8540, Japan;
  2. 2Department of Genetics, SOKENDAI, Mishima, Shizuoka 411-8540, Japan
    • 4 Present addresses: Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku Kobe 657-8501, Japan;

    • 5 Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    1. 3 These authors contributed equally to this work.

    Abstract

    Eukaryotic chromosomal DNA replication requires cyclin-dependent kinase (CDK) activity. CDK phosphorylates two yeast replication proteins, Sld3 and Sld2, both of which bind to Dpb11 when phosphorylated. These phosphorylation-dependent interactions are essential and are the minimal requirements for CDK-dependent activation of DNA replication. However, how these interactions activate DNA replication has not been elucidated. Here, we show that CDK promotes the formation of a newly identified fragile complex, the preloading complex (pre-LC) containing DNA polymerase ɛ (Pol ɛ), GINS, Sld2, and Dpb11. Formation of the pre-LC requires phosphorylation of Sld2 by CDK, but is independent of DNA replication, protein association with replication origins, and Dbf4-dependent Cdc7 kinase, which is also essential for the activation of DNA replication. We also demonstrate that Pol ɛ, GINS, Dpb11, and CDK-phosphorylated Sld2 form a complex in vitro. The genetic interactions between Pol ɛ, GINS, Sld2, and Dpb11 suggest further that they form an essential complex in cells. We propose that CDK regulates the initiation of DNA replication in budding yeast through formation of the pre-LC.

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