Regulated eukaryotic DNA replication origin firing with purified proteins

Nature. 2015 Mar 26;519(7544):431-5. doi: 10.1038/nature14285. Epub 2015 Mar 4.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / metabolism
  • Cyclin-Dependent Kinases / metabolism
  • DNA Replication*
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase / metabolism
  • Minichromosome Maintenance Proteins / metabolism
  • Multienzyme Complexes / metabolism
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism
  • Replication Origin / genetics
  • Replication Origin / physiology*
  • Replication Protein A / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / isolation & purification*
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • CDC45 protein, S cerevisiae
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Multienzyme Complexes
  • Multiprotein Complexes
  • Nuclear Proteins
  • RFA1 protein, S cerevisiae
  • Replication Protein A
  • Saccharomyces cerevisiae Proteins
  • CDC7 protein, S cerevisiae
  • Protein-Serine-Threonine Kinases
  • Cyclin-Dependent Kinases
  • DNA synthesome
  • DNA-Directed DNA Polymerase
  • Minichromosome Maintenance Proteins