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Mutational analysis of conserved aspartic acid residues in the Methanothermobacter thermautotrophicus MCM helicase

Nozomi Sakakibara1, Rajesh Kasiviswanathan, Zvi Kelman

  • 1Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA. sakakibaran@niaid.nih.gov

Extremophiles : Life Under Extreme Conditions
|January 29, 2011
PubMed

Insights

Conserved Asp residues in archaeal minichromosome maintenance (MCM) helicases are crucial for DNA binding and stability. ATP can rescue DNA binding deficiencies in mutant MCM proteins, suggesting a regulatory role.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Minichromosome maintenance (MCM) helicases are essential replicative enzymes in archaea and eukarya.
  • The archaeal MCM helicase has N-terminal, catalytic, and C-terminal regions, with the N-terminal domain involved in DNA binding and multimerization.
  • Specific conserved Asp residues in domain C of the N-terminal region are implicated in MCM function.

Purpose of the Study:

  • To investigate the role of conserved Asp residues (Asp202 and Asp203) in the N-terminal domain of the Methanothermobacter thermautotrophicus MCM helicase.
  • To determine the impact of mutations in these residues on DNA binding, thermal stability, and enzymatic activities.
  • To explore the potential regulatory role of ATP in MCM DNA binding.

Main Methods:

  • Site-directed mutagenesis of conserved Asp residues to Asn in the M. thermautotrophicus MCM protein.
  • DNA binding assays to assess the affinity of wild-type and mutant MCM proteins for DNA.
  • Thermal stability assays to evaluate the structural integrity of the proteins.
  • Helicase and ATPase activity assays to measure enzymatic function.

Main Results:

  • Mutations of Asp202 and Asp203 to Asn significantly reduced DNA binding ability and thermal stability of the MCM protein.
  • Despite impaired DNA binding, the mutant MCM proteins retained helicase and ATPase activities.
  • The presence of ATP was found to rescue the DNA binding deficiencies in the mutant MCM proteins.

Conclusions:

  • The conserved Asp residues in the N-terminal domain of archaeal MCM helicases play a critical role in DNA binding and protein stability.
  • ATP binding appears to modulate the DNA binding capability of the MCM helicase, suggesting a regulatory mechanism.
  • These findings provide insights into the structure-function relationships of MCM helicases and their role in DNA replication.

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