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Differences in the single-stranded DNA binding activities of MCM2-7 and MCM467: MCM2 and MCM5 define a slow

Matthew L Bochman1, Anthony Schwacha1

  • 1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.

The Journal of Biological Chemistry
|September 27, 2007
PubMed

Insights

The MCM2-7 complex, the eukaryotic replicative DNA helicase, lacks in vitro helicase activity. Differences in DNA binding and ATP-dependent association rates between MCM2-7 and the active MCM467 subcomplex suggest regulatory roles for MCM2/5 subunits.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The MCM2-7 complex is the proposed eukaryotic replicative DNA helicase.
  • Only the MCM467 subcomplex, not the full MCM2-7 complex, shows in vitro helicase activity.
  • The function of MCM2, MCM3, and MCM5 subunits in the MCM2-7 complex remains unclear.

Purpose of the Study:

  • To investigate why the MCM2-7 complex lacks in vitro helicase activity.
  • To compare the biochemical properties of MCM2-7 and MCM467 complexes.
  • To elucidate the role of MCM2, MCM3, and MCM5 subunits.

Main Methods:

  • Biochemical characterization of Saccharomyces cerevisiae MCM2-7 and MCM467 complexes.
  • Assays for DNA binding (ssDNA and dsDNA) and ATP-dependent activity.
  • Comparative analysis of complex association rates with ssDNA.

Main Results:

  • Both MCM2-7 and MCM467 complexes exhibit toroidal structure and ATP-dependent ssDNA binding.
  • MCM467 demonstrates superior dsDNA binding compared to MCM2-7.
  • MCM2-7 shows a slower ssDNA association rate, which is enhanced by ATP preincubation or MCM2/5 active site mutations.

Conclusions:

  • The lack of MCM2-7 helicase activity is not due to impaired ssDNA binding.
  • Differences in DNA binding and association kinetics suggest a regulatory role for MCM2/5 subunits.
  • Conformational changes at the MCM2/5 active site likely influence MCM2-7 complex activity and regulation.

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