The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum delta H minichromosome maintenance protein.
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 recombinant M. thermoautotrophicum MCM protein (MtMCM) in both Escherichia 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 in M. thermoautotrophicum. This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative helicase has yet to be identified.
Shechter
D F
DF
Department of Genetics and Development, Columbia University, New York, New York 10032, USA.
Ying
C Y
CY
Gautier
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019843:Archaeal Proteins / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004926:Escherichia coli; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D017014:Methanobacterium / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection
D019843:Archaeal Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C403162:MCM protein, Methanobacterium thermoautotrophicum; D008239:Lysine