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J Biol Chem, Vol. 275, Issue 20, 15049-15059, May 19, 2000
From the Department of Genetics and Development, the Department of
Dermatology, and the 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'
The Intrinsic DNA Helicase Activity of Methanobacterium
thermoautotrophicum
H Minichromosome Maintenance Protein*
,§, and Integrated Program in Cellular,
Molecular and Biophysical Studies, Columbia University,
New York, New York 10032
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.
*
This work was supported in part by American Cancer Society
Grant RPG-99-040-01-CCG (to J. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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