Eukaryotic Origin-Dependent DNA Replication in Vitro Reveals Sequential Action of DDK and S-CDK Kinases

Cell. 2011 Jul 8;146(1):80-91. doi: 10.1016/j.cell.2011.06.012.

Abstract

Proper eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) but not S phase cyclin-dependent kinase (S-CDK) is required for the initial origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Likewise, in vivo, DDK drives early-firing-origin recruitment of Cdc45 before activation of S-CDK. After S-CDK activation, a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Finally, recruitment of lagging but not leading strand DNA polymerases depends on Mcm10 and DNA unwinding. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to origin DNA unwinding and RNA primer synthesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / metabolism*
  • Cyclin-Dependent Kinases / metabolism*
  • DNA Polymerase I / metabolism
  • DNA Replication*
  • DNA-Binding Proteins / metabolism
  • G1 Phase
  • Nuclear Proteins / metabolism
  • Protein-Serine-Threonine Kinases / metabolism*
  • Replication Origin
  • S Phase
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • CDC45 protein, S cerevisiae
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Saccharomyces cerevisiae Proteins
  • CDC7 protein, S cerevisiae
  • Protein-Serine-Threonine Kinases
  • Cyclin-Dependent Kinases
  • DNA Polymerase I

Associated data

  • GEO/GSE29646