Concerted Loading of Mcm2-7 Double Hexamers Around DNA During DNA Replication Origin Licensing

Cell. 2009 Nov 13;139(4):719-30. doi: 10.1016/j.cell.2009.10.015. Epub 2009 Nov 5.

Abstract

The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1*Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication.

Publication types

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

MeSH terms

  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / isolation & purification
  • Cell Cycle Proteins / metabolism*
  • Chromosomal Proteins, Non-Histone / chemistry
  • Chromosomal Proteins, Non-Histone / isolation & purification
  • Chromosomal Proteins, Non-Histone / metabolism*
  • DNA / metabolism*
  • DNA Helicases / metabolism
  • DNA Replication
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Minichromosome Maintenance Complex Component 3
  • Minichromosome Maintenance Complex Component 4
  • Minichromosome Maintenance Complex Component 6
  • Minichromosome Maintenance Complex Component 7
  • Models, Molecular
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / isolation & purification
  • Nuclear Proteins / metabolism*
  • Origin Recognition Complex / metabolism
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • CDC6 protein, S cerevisiae
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • MCM5 protein, S cerevisiae
  • Nuclear Proteins
  • ORC1 protein, S cerevisiae
  • Origin Recognition Complex
  • Saccharomyces cerevisiae Proteins
  • DNA
  • DNA Helicases
  • MCM2 protein, S cerevisiae
  • MCM3 protein, S cerevisiae
  • MCM4 protein, S cerevisiae
  • MCM6 protein, S cerevisiae
  • MCM7 protein, S cerevisiae
  • Minichromosome Maintenance Complex Component 3
  • Minichromosome Maintenance Complex Component 4
  • Minichromosome Maintenance Complex Component 6
  • Minichromosome Maintenance Complex Component 7