Identification of amino acids important for the biochemical activity of Methanothermobacter thermautotrophicus MCM.

Methanothermobacter thermautotrophicus minichromosomal maintenance protein (mtMCM) is a 75 kDa protein that self-assembles into a double hexamer structure. The double hexamer formed by the N-terminal region of mtMCM has a highly charged (overwhelmingly net positive) inner channel. Here we investigate the effects of point mutations of some of these charged residues on the biological activities of mtMCM. Although all of the mutants were similar to the wild type in protein folding and complex assembly, we found that mutations impaired helicase activity. The study of the DNA binding and ATPase activities of these mutants revealed that the impairment of the helicase activity was highly correlated with a decrease in DNA binding, providing evidence consistent with the role of these charged residues of the inner channel in interactions with DNA.

Biochemical activities of the BOB1 mutant in Methanobacterium thermoautotrophicum MCM.

Minichromosomal maintenance proteins (MCMs) are considered to be the replicative helicase. Methanobacterium thermoautotrophicum has a single MCM gene (mtMCM). The crystal structure of the mtMCM N-terminal region is a double hexamer. Structure-guided sequence alignment indicates a structural conservation of this fragment across archaeal and eukaryotic MCMs. The mtMCM structure was successfully used to analyze a Saccharomyces cerevisiae MCM5 mutant, called BOB1, which contains a single residue change from Pro to Leu and bypasses a kinase normally required for initiation of DNA replication. A domain-push model was proposed to explain the BOB1 bypass activity. Here we investigate the effects of BOB1 mutation on the biochemical activities of mtMCM. Surprisingly, the BOB1 mutation (P62L) had a major effect on the helicase activity but had no significant impact on DNA binding and ATPase activities. These results will contribute to a more detailed understanding of the BOB1 bypass activity and other aspects of DNA replication control.

Double hexamer disruption and biochemical activities of Methanobacterium thermoautotrophicum MCM.

Methanobacterium thermoautotrophicum MCM (mtMCM) is a helicase required for DNA replication. Previous electron microscopy studies have shown mtMCM in several oligomeric forms. However, biochemical studies suggest that mtMCM is a dodecamer, likely a double hexamer (dHex). The crystal structure of the N-terminal fragment of mtMCM reveals a stable dHex architecture. To further confirm that the dHex is not an artifact of crystal packing of two hexamers, we investigated the relevance of the dHex by disrupting the hexamer-hexamer interactions seen in the crystal structure via site-directed mutagenesis and examining various biochemical activities of the mutants in vitro. Using a combination of biochemical and structural assays, we demonstrated that changing arginine to alanine at amino acid position 161 or the insertion of a six-aminoacid peptide at the hexamer-hexamer interface disrupted dHex formation and produced stable single hexamers (sHex). Furthermore, we showed that the sHex mutants retained wild-type level of ATPase and DNA binding activities but had decreased helicase activity when compared with the wild type dHex protein. These biochemical properties of mtMCM are reminiscent of those of SV40 large T antigen, suggesting that the dHex form of mtMCM may be the active helicase for DNA unwinding during the bidirectional DNA replication.

Polymorphism and double hexamer structure in the archaeal minichromosome maintenance (MCM) helicase from Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum minichromosome maintenance complex (mtMCM), a cellular replicative helicase, is a useful model for the more complex eukaryotic MCMs. Biochemical and crystallographic evidence indicates that mtMCM assembles as a double hexamer (dHex), but previous electron microscopy studies reported only the presence of single heptamers or single hexamers (Pape, T., Meka, H., Chen, S., Vicentini, G., Van Heel, M., and Onesti, S. (2003) EMBO Rep. 4, 1079-1083; Yu, X., VanLoock, M. S., Poplawski, A., Kelman, Z., Xiang, T., Tye, B. K., and Egelman, E. H. (2002) EMBO Rep. 3, 792-797). Here we present the first three-dimensional electron microscopy reconstruction of the full-length mtMCM dHex in which two hexamers contact each other via the structurally well defined N-terminal domains. The dHex has obvious side openings that resemble the side channels of LTag (large T antigen). 6-fold and 7-fold rings were observed in the same mtMCM preparation, but we determined that assembly as a double ring favors 6-fold structures. Additionally, open rings were also detected, which suggests a direct mtMCM loading mechanism onto DNA.

The structure and function of MCM from archaeal M. Thermoautotrophicum.

Eukaryotic chromosomal DNA is licensed for replication precisely once in each cell cycle. The mini-chromosome maintenance (MCM) complex plays a role in this replication licensing. We have determined the structure of a fragment of MCM from Methanobacterium thermoautotrophicum (mtMCM), a model system for eukaryotic MCM. The structure reveals a novel dodecameric architecture with a remarkably long central channel. The channel surface has an unusually high positive charge and binds DNA. We also show that the structure of the N-terminal fragment is conserved for all MCMs proteins despite highly divergent sequences, suggesting a common architecture for a similar task: gripping/remodeling DNA and regulating MCM activity. An mtMCM mutant protein equivalent to a yeast MCM5 (CDC46) protein with the bob1 mutation at its N terminus has only subtle structural changes, suggesting a Cdc7-bypass mechanism by Bob1 in yeast. Yeast bypass experiments using MCM5 mutant proteins support the hypothesis for the bypass mechanism.