Cdc6/Orc1 from Pyrococcus furiosus may act as the origin recognition protein and Mcm helicase recruiter.

Archaea have one or more Cdc6/Orc1 proteins, which share sequence similarities with eukaryotic Cdc6 and Orc1. These proteins are involved in the initiation process of DNA replication, although their specific function has not been elucidated, except for origin recognition and binding. We showed that the Cdc6/Orc1 protein from the hyperthermophilic archaeon Pyrococcus furiosus specifically binds to the oriC region in the whole genome. However, it remains unclear how this initiator protein specifically recognizes the oriC region and how the Mcm helicase is recruited to oriC. In the current study, we characterized the biochemical properties of Cdc6/Orc1 in P. furiosus. The ATPase activity of the Cdc6/Orc1 protein was completely suppressed by binding to DNA containing the origin recognition box (ORB). Limited proteolysis and DNase I-footprint experiments suggested that the Cdc6/Orc1 protein changes its conformation on the ORB sequence in the presence of ATP. This conformational change may have an unknown, important function in the initiation process. Results from an in vitro recruiting assay indicated that Mcm is recruited onto the oriC region in a Cdc6/Orc1-dependent, but not ATP-dependent, manner. However, some other function is required for the functional loading of this helicase to start the unwinding of the replication fork DNA.

Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus.

The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.

Schizosaccharomyces pombe Orc5 plays multiple roles in the maintenance of genome stability throughout the cell cycle.

The six-subunit origin recognition complex (ORC) acts as a landing pad for factors that initiate DNA replication by binding to replication origins. In addition, ORC is involved in other processes such as transcriptional gene silencing and sister chromatid cohesion in Saccharomyces cerevisiae. However, whether these functions of ORC are specific to Saccharomyces cerevisiae or are shared by the ORC of other organisms is currently unclear. Analysis of two temperature-sensitive alleles of the fifth ORC subunit of Schizosaccharomyces pombe, orc5-H19 and orc5-H37, indicates that Orc5 of Schizosaccharomyces pombe has similar multiple functions to those of Orc5 of Saccharomyces cerevisiae. The orc5-H19 cells were defective in DNA replication initiation, and execution point analysis of this mutant revealed that ORC functions before metaphase to prepare for the initiation of replication in the next cell cycle. The orc5-H37 cells seemed to complete DNA synthesis but were arrested before entering M phase. In both mutants, the rads-chk1 checkpoint was activated to prevent mitosis, suggesting that this checkpoint pathway monitors the functional integrity of ORC. In addition, orc5-H37 cells showed premature separation of sister chromatids, which resulted in cell growth being dependent on the mad2-dependent spindle checkpoint. Consistently, this mutant showed a defect in the loading of Rad21, a cohesin component. Based on these observations, we propose that Orc5 has at least two distinct functions that can be separated genetically. Taken together, our results provide further support for the idea that ORC plays multiple functions during the cell cycle.

The GINS complex from Pyrococcus furiosus stimulates the MCM helicase activity.

Pyrococcus furiosus, a hyperthermophilic Archaea, has homologs of the eukaryotic MCM (mini-chromosome maintenance) helicase and GINS complex. The MCM and GINS proteins are both essential factors to initiate DNA replication in eukaryotic cells. Many biochemical characterizations of the replication-related proteins have been reported, but it has not been proved that the homologs of each protein are also essential for replication in archaeal cells. Here, we demonstrated that the P. furiosus GINS complex interacts with P. furiosus MCM. A chromatin immunoprecipitation assay revealed that the GINS complex is detected preferentially at the oriC region on Pyrococcus chromosomal DNA during the exponential growth phase but not in the stationary phase. Furthermore, the GINS complex stimulates both the ATPase and DNA helicase activities of MCM in vitro. These results strongly suggest that the archaeal GINS is involved in both the initiation and elongation processes of DNA replication in P. furiosus, as observed in eukaryotic cells.

Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.

The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation.

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin.

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.