In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization

Nucleic Acids Res. 2013 Mar 1;41(5):3162-72. doi: 10.1093/nar/gkt043. Epub 2013 Feb 1.

Abstract

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2-7 onto DNA. Helicase loading involves two MCM2-7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2-7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC-Cdc6 interaction and MCM2-7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2-7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2-7. To determine whether Cdc6 regulates MCM2-7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2-7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2-7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2-7 recruitment, show that ATPase activity is required for MCM2-7 hexamer dimerization and demonstrate that MCM2-7 hexamers are recruited to origins in a consecutive process.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / chemistry
  • Amino Acid Substitution
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / genetics
  • Cell Line
  • Chromosomal Proteins, Non-Histone / chemistry
  • DNA Replication
  • DNA, Fungal / chemistry
  • DNA-Binding Proteins / chemistry
  • Hydrolysis
  • Minichromosome Maintenance Complex Component 7
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / chemistry
  • Origin Recognition Complex / antagonists & inhibitors
  • Origin Recognition Complex / chemistry
  • Origin Recognition Complex / genetics
  • Protein Binding
  • Protein Multimerization*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / antagonists & inhibitors
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics

Substances

  • CDC6 protein, S cerevisiae
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • DNA, Fungal
  • DNA-Binding Proteins
  • Nuclear Proteins
  • ORC1 protein, S cerevisiae
  • ORC4 protein, S cerevisiae
  • Origin Recognition Complex
  • Saccharomyces cerevisiae Proteins
  • TAH11 protein, S cerevisiae
  • adenosine 5'-O-(3-thiotriphosphate)
  • Adenosine Triphosphate
  • MCM2 protein, S cerevisiae
  • MCM7 protein, S cerevisiae
  • Minichromosome Maintenance Complex Component 7