title: Mutational analysis of conserved aspartic acid residues in the Methanothermobacter thermautotrophicus MCM helicase.
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.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA. sakakibaran@niaid.nih.gov
authors.forename: Nozomi
authors.initials: N
authors.lastname: Sakakibara
authors.forename: Rajesh
authors.initials: R
authors.lastname: Kasiviswanathan
authors.forename: Zvi
authors.initials: Z
authors.lastname: Kelman
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002850:Chromatography, Gel; D002942:Circular Dichroism / Q000379:methods; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry*; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000235:genetics; D017013:Methanobacteriaceae / Q000201:enzymology*; D026341:Minichromosome Maintenance 1 Protein / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
chemical_list: D004277:DNA, Single-Stranded; D026341:Minichromosome Maintenance 1 Protein; D001224:Aspartic Acid; D004247:DNA; D004265:DNA Helicases