ignificant helicase activity in vitro . However, the DNA helicase activity was clearly stimulated by the addition of GINS (the Gins23-Gins51 complex), which is the homolog of the eukaryotic GINS complex (described below in more detail). This result suggests that MCM works with other accessory factors to form a core complex in P. furiosus similar to the eukaryotic CMG complex as described above. archaeal organisms have more than two Cdc6/Orc1 homologs. It was found that the two Cdc6/Orc1 homologs, Cdc6-1 and Cdc6-2, both inhibit the helicase activity of MCM in M. thermautotrophicus . Similarly, Cdc6-1 inhibits MCM activity in S. solfataricus . In contrast, the Cdc6-2 protein stimulates the helicase activity of MCM in Thermoplasma acidophilum . Functional interactions between Cdc6/Orc1 and Mcm proteins need to be investigated in greater detail to achieve a more comprehensive understanding of the conservation and diversity of the initiation mechanism in archaeal DNA replication. interesting feature of DNA replication initiation is that several archaea have multiple genes encoding Mcm homologs in their genomes. Based on the recent comprehensive genomic analyses, thirteen archaeal species have more than one mcm gene. However, many of the mcm genes in the archaeal genomes seem to reside within mobile elements, originating from viruses. For example, two of the three genes in the Thermococcus kodakarensis genome are located in regions where genetic elements have presumably been integrated. establishment of a genetic manipulation system for T. kodakarensis , is the first for a hyperthermophilic euryarchaeon, and is advantageous for investigating the function of these Mcm proteins. Two groups have recently performed gene disruption experiments for each mcm gene. These experiments revealed that the knock-out strains for mcm1 and mcm2 were easily isolated, but mcm3 could not be disrupted. Mcm3 is relatively abundant in the T. kodakarensis cells. Furthermore, an in vitro experiment using purified Mcm proteins showed that only Mcm3 forms a stable hexameric structure in solution. These results support the contention that Mcm3 is the main helicase core protein in the normal DNA replication process in T. kodakarensis . functions of the other two Mcm proteins remain to be elucidated. The genes for Mcm1 and Mcm2 are stably inherited, and their gene products may perform some important functions in the DNA metabolism in T. kodakarensis. The DNA helicase activity of the recombinant Mcm1 protein is strong in vitro, and a distinct amount of the Mcm1 protein is present in T . kodakarensis cells. Moreover, Mcm1 functionally interacts with the GINS complex from T. kodakarensis. These observations strongly suggest that Mcm1 does participate in some aspect of DNA transactions, and may be substituted with Mcm3. immunoprecipitation experiments showed that Mcm1 co-precipitated with Mcm3 and GINS, although they did not form a heterohexameric complex, ...
