The Intrinsic DNA Helicase Activity of Methanobacterium thermoautotrophicum ΔH Minichromosome Maintenance Protein

• Published in: The Journal of biological chemistry Vol. 275; no. 20; pp. 15049 - 15059
• Main Authors: Shechter, David F., Ying, Carol Y., Gautier, Jean
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19-05-2000
• Subjects: Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Animals; Archaeal Proteins - metabolism; atrial natriuretic hormone; Baculovirus; Binding Sites; Cell Line; Cloning, Molecular; Conserved Sequence; DNA Helicases - metabolism; DNA Replication; Escherichia coli; Kinetics; Lysine; Macromolecular Substances; Methanobacterium - chemistry; Methanobacterium - genetics; Methanobacterium - metabolism; Methanobacterium thermoautotrophicum; minichromosome maintenance proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Spodoptera; Substrate Specificity; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M000398200

Minichromosome maintenance proteins (MCMs) form a family of conserved molecules that are essential for initiation of DNA replication. All eukaryotes contain six orthologous MCM proteins that function as heteromultimeric complexes. The sequencing of the complete genomes of several archaebacteria has shown that MCM proteins are also present in archaea. The archaea Methanobacterium thermoautotrophicum contains a single MCM-related sequence. Here we report on the expression and purification of the recombinantM. thermoautotrophicum MCM protein (MtMCM) in bothEscherichia coli and baculovirus-infected cells. We show that purified MtMCM protein assembles in large macromolecular complexes consistent in size with being double hexamers. We demonstrate that MtMCM contains helicase activity that preferentially uses dATP and DNA-dependent dATPase and ATPase activities. The intrinsic helicase activity of MtMCM is abolished when a conserved lysine in the helicase domain I/nucleotide binding site is mutated. MtMCM helicase unwinds DNA duplexes in a 3′ → 5′ direction and can unwind up to 500 base pairs in vitro. The kinetics, processivity, and directionality of MtMCM support its role as a replicative helicase inM. thermoautotrophicum. This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative helicase has yet to be identified.