suggesting that Mcm1 is involved in the replisome or repairsome and shares some function in T. kodakarensis cells. Although western blot analysis could not detect Mcm2 in the extract from exponentially growing T. kodakarensis cells, a RT-PCR experiment detected the transcript of the mcm2 gene in the cells (Ishino et al., unpublished). The recombinant Mcm2 protein also has ATPase and helicase activities in vitro. Therefore, the mcm2 gene is expressed under normal growth conditions and may work in some process with a rapid turnover. Further experiments to measure the efficiency of mcm2 gene transcription by quantitative PCR, as well as to assess the stability of the Mcm2 protein in the cell extract, are needed. analyses investigating the sensitivities of the? mcm1 and? mcm2 mutant strains to DNA damage caused by various mutagens, as reported for other DNA repair-related genes in T. kodakarensis , may provide a clue to elucidate the functions of these Mcm proteins. Methanococcus maripaludis S2 harbors four mcm genes in its genome, three of which seem to be derived from phage, a shotgun proteomics study detected peptides originating from three out of the four mcm gene products. Furthermore, the four gene products co-expressed in E. coli cells were co-purified in the same fraction. These results suggest that multiple Mcm proteins are functional in the M. maripaludis cells.
5. Recruitment of Mcm to the oriC region
important question is how MCM is recruited onto the unwound region of oriC. The detailed loading mechanism of the MCM helicase has not been elucidated. It is believed that archaea utilize divergent mechanisms of MCM helicase assembly at the oriC. in vitro recruiting assay showed that P. furiosus MCM is recruited to the oriC DNA in a Cdc6/Orc1-dependent manner. This assay revealed that preloading Cdc6/Orc1 onto the ORB DNA resulted in a clear reduction in MCM recruitment to the oriC region, suggesting that free Cdc6/Orc1 is preferable as a helicase recruiter, to associate with MCM and bring it to oriC. It would be interesting to understand how the two tasks, origin recognition and MCM recruiting, are performed by the Cdc6/Orc1 protein, because the WH domain, which primarily recognizes and binds ORB, also has strong affinity for the Mcm protein. assembly of the Mcm protein onto the ORB DNA by the Walker A-motif mutant of P. furiosus Cdc6/Orc1 occurred with the same efficiency as the wild type Cdc6/Orc1. The DNA binding of P. furiosus Cdc6/Orc1 was not drastically different in the presence and absence of ATP, as in the case of the initiator proteins from Archaeoglobus fulgidus , S. solfataricus , and A. pernix . Therefore, it is still not known whether the ATP binding and hydrolysis activity of Cdc6/Orc1 regulates the Mcm protein recruitment onto oriC in the cells. more important issue is the very low efficiency of the Mcm protein recruitment in the ...
