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

• Published in: The Journal of biological chemistry Vol. 275; no. 20; p. 15049
• Main Authors: David F. Shechter, Carol Y. Ying, Jean Gautier
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 19-05-2000
• 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 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.