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ATP binding and hydrolysis by Mcm2 regulate DNA binding by Mcm complexes

Brent E Stead1, Catherine D Sorbara, Christopher J Brandl

  • 1Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, Ontario, Canada.

Journal of Molecular Biology
|June 23, 2009
PubMed

Insights

Minichromosome maintenance (Mcm) proteins regulate DNA replication. Nucleotide binding by Mcm2 is critical for controlling the DNA binding and unwinding activities of Mcm complexes, impacting yeast viability.

Area of Science:

  • Molecular Biology
  • Eukaryotic DNA Replication
  • Protein Biochemistry

Background:

  • The minichromosome maintenance (Mcm) complex (Mcm2-7) is the core eukaryotic replicative helicase.
  • Subcomplexes, such as Mcm4/6/7, are capable of DNA unwinding.
  • Previous research indicated Mcm2 inhibits Mcm4/6/7 DNA unwinding activity.

Purpose of the Study:

  • To investigate the role of nucleotide binding by Saccharomyces cerevisiae Mcm2 in regulating Mcm complex DNA binding and unwinding.
  • To determine if Mcm2 requires nucleotide binding for its inhibitory function on Mcm4/6/7.

Main Methods:

  • Utilized an Mcm4/6/7 subcomplex as a tool to study Mcm2 regulation.
  • Employed an Mcm2 mutant defective for ATP hydrolysis (K549A) and ATP analogues.
  • Assessed DNA binding and unwinding activities of Mcm complexes under various nucleotide conditions.

Main Results:

  • Mcm2 interaction with Mcm4/6/7 is insufficient for inhibition; nucleotide binding by Mcm2 is required.
  • ADP binding to Mcm2 is necessary to inhibit DNA binding and unwinding by Mcm4/6/7, independent of Mcm3/5.
  • The Mcm2(K549A) mutation significantly altered DNA binding properties of the Mcm2-7 complex and impaired yeast viability.

Conclusions:

  • The nucleotide-bound state of Mcm2 is critical for regulating the DNA binding and unwinding activities of Mcm4/6/7 and Mcm2-7 complexes.
  • ATP hydrolysis by Mcm2 plays a vital role in the regulation of native Mcm complexes.
  • Mcm2's nucleotide-dependent regulation is essential for yeast viability.

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