in vivo replication initiation point (RIP) assay. Although the details of the replication machinery that must be established at the unwound site are not fully understood in Archaea, it is expected to minimally include MCM, GINS, primase, PCNA, DNA polymerase, and RPA, as described below. The following P. furiosus studies revealed that the ATPase activity of the Cdc6/Orc1 was completely suppressed by binding to DNA containing the ORB. proteolysis and DNase I-footprint experiments suggested that the Cdc6/Orc1 protein changes its conformation on the ORB sequence in the presence of ATP. The physiological meaning of this conformational change has not been solved, but it should have an important function to start the initiation process as in the case of bacterial DnaA protein. In addition, results from an in vitro recruiting assay indicated that MCM (Mcm protein complex), the replicative DNA helicase, is recruited onto the oriC region in a Cdc6/Orc1-dependent, but not ATP-dependent, manner, as described below. However, this recruitment is not sufficient for the unwinding function of MCM, and some other function remains to be identified for the functional loading of this helicase to promote the progression of the DNA replication fork.
4. MCM helicase
unwinding of the oriC region, the replicative helicase needs to remain loaded to provide continuous unwinding of double stranded DNA (dsDNA) as the replication forks progress bidirectionally . The MCM protein complex, consisting of six subunits (Mcm2, 3, 4, 5, 6, and 7), is known to be the replicative helicase core in eukaryotic cells. MCM further interacts with Cdc45 and GINS, to form a ternary assembly referred to as the CMG complex, that is believed to be the functional helicase in eukaryotic cells (Figure 3). However, this idea is still not universal for the eukaryotic replicative helicase.
Figure 3. DNA-Unwinding complex in eukaryotes and archaea.
The CMG complex is the replicative helicase for the template DNA unwinding reaction in eukaryotes. The archaeal genomes contain the homologs of the Mcm and Gins proteins, but a Cdc45 homolog has not been identified. Recent research suggests that a RecJ-like exonuclease GAN, which has weak sequence homology to that of Cdc45, may work as a helicase complex with MCM and GINS. archaeal genomes appear to encode at least one Mcm homologue, and the helicase activities of these proteins from several archaeal organisms have been confirmed in vitro . In contrast to the eukaryotic MCM, the archaeal MCMs, consist of a homohexamer or homo double hexamer, having distinct DNA helicase activity by themselves in vitro , and therefore, these MCMs on their own may function as the replicative helicase in vivo . structure-function relationships of the archaeal Mcms have been aggressively studied using purified proteins and site-directed mutagenesis. An early report using the ChIP method showed that the P. abyssi Mcm protein preferentially binds to the origin in vivo in exponentially growing cells. The P. furiosus MCM helicase does not display s...
