The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum Delta H minichromosome maintenance protein

Minichromosome maintenance proteins (MCMs) form a family of conserved molec ules that are essential for initiation of DNA replication. All eukaryotes c ontain six orthologous MCM proteins that function as heteromultimeric compl exes. The sequencing of the complete genomes of several archaebacteria has shown that MCM proteins are also present in archaea. The archaea Methanobac terium thermoautotrophicum contains a single MCM-related sequence. Here we report on the expression and purification of the recombinant M. thermoautot rophicum MCM protein (MtMCM) in both Escherichia coil and baculovirus-infec ted cells. We show that purified MtMCM protein assembles in large macromole cular complexes consistent in size with being double hexamers. We demonstra te that MtMCM contains helicase activity that preferentially uses dATP and DNA-dependent dATPase and ATPase activities. The intrinsic helicase activit y of MtMCM is abolished when a conserved lysine in the helicase domain I/nu cleotide 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 kinet ics, processivity, and directionality of MtMCM support its role as a replic ative helicase in M. thermoautotrophicum. This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative he licase has yet to be identified.