Mutational analysis of conserved aspartic acid residues in the <Emphasis Type="Italic">Methanothermobacter thermautotrophicus</Emphasis> MCM helicase |
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Authors: | Email author" target="_blank">Nozomi?SakakibaraEmail author Rajesh?Kasiviswanathan Zvi?Kelman |
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Institution: | (1) Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA;(2) Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, MD 20850, USA;(3) Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA;(4) Present address: Laboratory of Viral Diseases, NIAID, NIH, 4 Center Drive, Bethesda, MD 20892, USA;(5) Present address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, 111 TW Alexander Dr., Research Triangle Park, NC 27709, USA |
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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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