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Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement, and ATP site architecture

Megan J Davey1, Chiara Indiani, Mike O'Donnell

  • 1Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021, USA.

The Journal of Biological Chemistry
|December 14, 2002
PubMed

Insights

The Saccharomyces cerevisiae Mcm2-7p complex, essential for DNA replication, requires specific subunit pairs for ATP hydrolysis. This study identifies these pairs and predicts the heterohexamer

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Eukaryotic DNA Replication

Background:

  • The Mcm2-7p complex functions as the primary replicative helicase in eukaryotes.
  • All six subunits of the Mcm2-7p complex are crucial for DNA replication.
  • Understanding the Mcm2-7p complex's mechanism is vital for comprehending cell division.

Purpose of the Study:

  • To reconstitute the Mcm2-7p complex from individual recombinant Saccharomyces cerevisiae MCM proteins.
  • To investigate the ATPase activity of the Mcm2-7p complex and identify subunits responsible for ATP hydrolysis.
  • To elucidate the subunit arrangement and functional interactions within the Mcm2-7p heterohexamer.

Main Methods:

  • Purification of six Saccharomyces cerevisiae MCM proteins as recombinant proteins in Escherichia coli.
  • Reconstitution of the Mcm2-7p complex from individual subunits.
  • Analysis of ATPase activity in pairwise mixtures of MCM proteins and site-directed mutagenesis studies.

Main Results:

  • No single MCM protein efficiently hydrolyzes ATP; activity requires specific pairs.
  • Only three out of fifteen possible pairwise MCM protein mixtures exhibit ATPase activity.
  • An essential arginine in Mcm3p is critical for ATP hydrolysis on Mcm7p, and other subunits are linked to this pair.
  • The study predicts which subunits bind ATP and their arrangement within the Mcm2-7p heterohexamer.

Conclusions:

  • The Mcm2-7p replicative helicase is a complex machinery where specific subunit interactions drive ATP hydrolysis.
  • The findings provide insights into the functional roles of individual MCM subunits and their arrangement in the heterohexamer.
  • This research contributes to a deeper understanding of the eukaryotic DNA replication mechanism.

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