Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation

EMBO J. 2016 May 2;35(9):961-73. doi: 10.15252/embj.201593552. Epub 2016 Feb 24.

Abstract

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.

Keywords: DDK; DNA replication initiation; Sld3.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / metabolism*
  • DNA Replication
  • DNA-Binding Proteins / metabolism*
  • Minichromosome Maintenance Complex Component 4 / metabolism*
  • Minichromosome Maintenance Complex Component 6 / metabolism*
  • Nuclear Proteins / metabolism
  • Phosphopeptides / metabolism*
  • Protein Binding
  • Protein-Serine-Threonine Kinases / metabolism*
  • Replication Origin*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • CDC45 protein, S cerevisiae
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Phosphopeptides
  • Saccharomyces cerevisiae Proteins
  • Sld3 protein, S cerevisiae
  • CDC7 protein, S cerevisiae
  • Protein-Serine-Threonine Kinases
  • MCM4 protein, S cerevisiae
  • MCM6 protein, S cerevisiae
  • Minichromosome Maintenance Complex Component 4
  • Minichromosome Maintenance Complex Component 6