Pre-initiation complex assembly functions as a molecular switch that splits the Mcm2-7 double hexamer

EMBO Rep. 2017 Oct;18(10):1752-1761. doi: 10.15252/embr.201744206. Epub 2017 Aug 17.

Abstract

Initiation of chromosomal DNA replication in eukaryotes involves two steps: licensing and firing. In licensing, a core component of the replicative helicase, the Mcm2-7 complex, is loaded onto replication origins as an inactive double hexamer, which is activated in the firing step by firing factors. A reaction intermediate called the pre-initiation complex (pre-IC) has been proposed to assemble transiently during firing, but the existence of the pre-IC has not yet been confirmed. Here, we show, by systematic chromatin immunoprecipitation, that a distinct intermediate that fits the definition of the pre-IC assembles during firing in the budding yeast Saccharomyces cerevisiae Pre-IC assembly is observed in the absence of Mcm10, one of the firing factors, and is mutually dependent on all the firing factors whose association to replication origins is triggered by cyclin-dependent kinase. In the pre-IC, the Mcm2-7 double hexamer is separated into single hexamers, as in the active helicase. Our data indicate that pre-IC assembly functions as an all-or-nothing molecular switch that splits the Mcm2-7 double hexamer.

Keywords: DNA polymerase ε; DNA replication; origin firing; pre‐IC (pre‐initiation complex); systematic ChIP.

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • DNA Replication*
  • DNA, Fungal / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Immunoprecipitation
  • Minichromosome Maintenance Proteins / chemistry*
  • Minichromosome Maintenance Proteins / genetics
  • Minichromosome Maintenance Proteins / metabolism*
  • Replication Origin
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / economics
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Cell Cycle Proteins
  • DNA, Fungal
  • DNA-Binding Proteins
  • MCM10 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Minichromosome Maintenance Proteins