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

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Abstract

Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the protein is divided into three domains, A, B, and C, and was shown to be involved in protein multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter thermautotrophicus MCM protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant proteins. Possible roles of these conserved residues in MCM function are discussed.

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Abbreviations

CD:

Circular dichroism

dsDNA:

Double-stranded DNA

DSC:

Differential scanning calorimetry

MCM:

Minichromosome maintenance

mt:

Methanothermobacter thermautotrophicus

ssDNA:

Single-stranded DNA

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Acknowledgments

We would like to thank Dr. Lori Kelman for her comments on the manuscript. Specialty oligonucleotide synthesis was supported as part of a NIST/UMD Joint Program in Bioscience Measurements (70NANB9H9196). This work was supported by a grant from the National Science Foundation (MCB-0815646).

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Author notes

  1. Nozomi Sakakibara

    Present address: Laboratory of Viral Diseases, NIAID, NIH, 4 Center Drive, Bethesda, MD, 20892, USA

  2. Rajesh Kasiviswanathan

    Present address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, 111 TW Alexander Dr., Research Triangle Park, NC, 27709, USA

Authors and Affiliations

  1. Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD, 20850, USA

    Nozomi Sakakibara & Rajesh Kasiviswanathan

  2. Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, MD, 20850, USA

    Zvi Kelman

  3. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA

    Zvi Kelman

Authors
  1. Nozomi Sakakibara
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  2. Rajesh Kasiviswanathan
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  3. Zvi Kelman
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Corresponding author

Correspondence to Nozomi Sakakibara.

Additional information

Communicated by L. Huang.

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Sakakibara, N., Kasiviswanathan, R. & Kelman, Z. Mutational analysis of conserved aspartic acid residues in the Methanothermobacter thermautotrophicus MCM helicase. Extremophiles 15, 245–252 (2011). https://doi.org/10.1007/s00792-010-0352-1

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  • DOI: https://doi.org/10.1007/s00792-010-0352-1

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