reported in vitro assay. Quantification of the recruited Mcm protein by the in vitro assay showed that less than one Mcm hexamer was recruited to the ORB. The linear DNA containing ORB1 and ORB2, used in the recruiting assay, may not be suitable to reconstitute the archaeal DNA replication machinery and a template that more closely mimics the chromosomal DNA may be required. , It may be that as yet unidentified proteins are required to achieve efficient in vitro helicase loading in the P. furiosus cells. Finally, it will ultimately be necessary to construct a more defined in vitro replication system to analyze the regulatory functions of Cdc6/Orc1 precisely during replication initiation. M. thermautotrophicus , the Cdc6-2 proteins can dissociate the Mcm multimers. The activity of Cdc6-2 might be required as the MCM helicase loader in this organism. The interaction between Cdc6/Orc1 and Mcm is probably general. However, the effect of Cdc6/Orc1 on the MCM helicase activity differs among various organisms, as described above. Some other protein factors may function in various archaea, for example a protein that is distantly related to eukaryotic Cdt1, which plays a crucial role during MCM loading in Eukaryota, exists in some archaeal organisms, although its function has not been characterized yet.
6. GINS
The eukaryotic GINS complex was originally identified in Saccharomyces cerevisiae as essential protein factor for the initiation of DNA replication. GINS consists of four different proteins, Sld5, Psf1, Psf2, and Psf3 (therefore, GINS is an acronym for Japanese go-ichi-ni-san, meaning 5-1-2-3, after these four subunits). The amino acid sequences of the four subunits in the GINS complex share some conservation, suggesting that they are ancestral paralogs. However, most of the archaeal genomes have only one gene encoding this family protein, and more interestingly, the Crenarchaeota and Euryarchaeota (the two major subdomains of Archaea) characteristically have two genes with sequences similar to Psf2 and Psf3, and Sld5 and Psf1, respectively referred to as Gins23 and Gins51. Gins homolog, designated as Gins23, was biochemically detected in S. solfataricus as the first Gins protein in Archaea, in a yeast two-hybrid screening for interaction partners of the Mcm protein, and another subunit, designated as Gins15, was identified by mass-spectrometry analysis of an immunoaffinity-purified native GINS from an S. solfataricus cell extract. The S. solfataricus GINS, composed of two proteins, Gins23 and Gins15, forms a tetrameric structure with a 2:2 molar ratio. The GINS from P. furiosus , a complex of Gins23 and Gins51 with a 2:2 ratio, was identified as the first euryarchaeal GINS. Gins51 was preferred over Gins15 because of the order of the name of GINS. MCM2-7 hexamer was copurified in complex with Cdc45 and GINS from Drosophila melanogaster embryo extracts and S. cerevisiae lysates, and the CMG (Cdc45-MCM2-7-GINS) complex (Figure 3), as described above, should be important for the function of the replicative helicase. The CMG com...
