The interaction between an origin sequence and the origin recognition complex (ORC), which is highly conserved in eukaryotes, is critical for the initiation of DNA replication. In this report, we have examined the interaction between the Schizosaccharomyces pombe (sp) autonomously replicating sequence 1 (ars1) and the spORC. For this purpose, we have purified the spORC containing all six subunits, a six-subunit complex containing the N-terminaldeleted spOrc4 subunit (spORC ), and the spOrc4 subunit by using the baculovirus expression system. Wild-type spORC showed sequence-specific binding to ars1, and the spOrc4 protein alone showed the same DNA-binding properties as wild-type spORC. In contrast, the spORC ⌬N-Orc4 and the ⌬N-spOrc4p alone did not bind significantly to ars1. These findings indicate that the N-terminal domain of the spOrc4 protein that contains multiple AT-hook motifs is essential for the ars1-binding activity. DNA-binding competition assays with fragments of ars1 and DNase I footprinting studies with full-length ars1 revealed that the spORC interacted with several AT-rich sequence regions of ars1. These DNA-binding properties of spORC correlate with the previously determined sequence requirements of the S. pombe ars1. These studies indicate that because of its unique Orc4 subunit, S. pombe uses a mechanism to recognize its origins different from that used by Saccharomyces cerevisiae. D NA replication origin sequences in Saccharomyces cerevisiaeare relatively short AT-rich sequences (100-150 bp) that include an essential autonomously replicating sequence (ARS) consensus sequence (ACS) (1-3). In contrast, replicators in higher eukaryotes are much longer and share no recognizable essential motifs (4, 5). The ARS elements of Schizosaccharomyces pombe are larger (longer than 700 bp) than those of Sac. cerevisiae, and contain several redundant AT-rich sequences that are important for ARS activity, but lack a conserved essential sequence motif such as an ACS (6-8).The origin recognition complex (ORC) was initially purified as an ARS-binding protein from Sac. cerevisiae (9). ORC consists of six distinct subunits, Orc1p to Orc6p, which are all essential for DNA replication (10-12). Sac. cerevisiae ORC specifically recognizes and binds to the ACS containing an 11-bp AT-rich sequence in vivo and in vitro, and ATP is essential for this ARS-binding activity (9,13,14). ORC binds to replication origin DNA throughout the cell cycle and is required to recruit other essential initiation factors for the assembly of the prereplicative complex (15,16). ORC is conserved in all eukaryotes and has been identified and purified from other species, including S. pombe, Drosophila melanogaster, Xenopus laevis, and humans (17)(18)(19)(20)(21)(22). Although in vivo and in vitro studies suggest that ORC is essential for DNA replication in these species (15,16), the interactions between these ORCs and their replication origin DNAs have not been characterized except for Sac. cerevisiae. D. melanogaster ORC interacts with multip...

The origin recognition complex (ORC), first identified in Saccharomyces cerevisiae (sc), is a six-subunit protein complex that binds to DNA origins. Here, we report the identification and cloning of cDNAs encoding the six subunits of the ORC of Schizosaccharomyces pombe (sp). Sequence analyses revealed that spOrc1, 2, and 5 subunits are highly conserved compared with their counterparts from S. cerevisiae, Xenopus, Drosophila, and human. In contrast, both spOrc3 and spOrc6 subunits are poorly conserved. As reported by Chuang and Kelly [(1999) Proc. Natl. Acad. Sci. USA 96, 2656 -2661], the C-terminal region of spOrc4 is also conserved whereas the N terminus uniquely contains repeats of a sequence that binds strongly to AT-rich DNA regions. Consistent with this, extraction of S. pombe chromatin with 1 M NaCl, or after DNase I treatment, yielded the six-subunit ORC, whereas extraction with 0.3 M resulted in five-subunit ORC lacking spOrc4p. The spORC can be reconstituted in vitro with all six recombinant subunits expressed in the rabbit reticulocyte system. The association of spOrc4p with the other subunits required the removal of DNA from reaction mixture by DNase I. This suggests that a strong interaction between spOrc4p and DNA can prevent the isolation of the six-subunit ORC. The unique DNA-binding properties of the spORC may contribute to our understanding of the sequence-specific recognition required for the initiation of DNA replication in S. pombe.replication ͉ AT-rich sequences I nitiation of eukaryotic DNA replication occurs at multiple sites distributed throughout the entire genome. The cis-acting elements that contribute to the firing of replication origins have been studied intensively in Saccharomyces cerevisiae. The conserved sequences that are required for DNA replication, called autonomous replication sequences (ARS elements), consist of an essential A domain that contains an 11-bp ARS consensus sequence (ACS) and B domains that include sequences that enhance replication (1-3). The identification of ACS in S. cerevisiae played an important role in the isolation of the six-subunit (Orc1p-Orc6p) origin recognition complex (ORC), which specifically binds to the ACS in vivo and in vitro (4-6). Biochemical and genetic studies have shown that the genes encoding the Orc subunits are essential and required for DNA replication (7,8). It has been shown that the ORC contains weak ATPase activity and that ATP is essential for its binding to ARS elements (9). Homologues of the ORC subunits have been found in a number of eukaryotes and in Drosophila melanogaster (dm) and Xenopus laevis (xl), These proteins have been shown to form a six-protein complex similar to that observed in S. cerevisiae (10-12). Furthermore, isolated xlORC preparations can restore the DNA replication activity of egg extracts of Xenopus depleted of the ORC proteins (11, 13). Similar experiments, carried out with recombinant dmORC proteins, can restore the DNA replication activity of egg extracts of both Xenopus and Drosophila (14). These re...

In both budding and fission yeasts, a null mutation of the DNA2 gene is lethal. In contrast, a null mutation of Caenorhabditis elegans dna2+ causes a delayed lethality, allowing survival of some mutant C.elegans adults to F2 generation. In order to understand reasons for this difference in requirement of Dna2 between these organisms, we examined the enzymatic properties of the recombinant C.elegans Dna2 (CeDna2) and its interaction with replication-protein A (RPA) from various sources. Like budding yeast Dna2, CeDna2 possesses DNA-dependent ATPase, helicase and endonuclease activities. The specific activities of both ATPase and endonuclease activities of the CeDna2 were considerably higher than the yeast Dna2 (∼10- and 20-fold, respectively). CeDna2 endonuclease efficiently degraded a short 5′ single-stranded DNA tail (<10 nt) that was hardly cleaved by ScDna2. Both endonuclease and helicase activities of CeDna2 were stimulated by CeRPA, but not by human or yeast RPA, demonstrating a species-specific interaction between Dna2 and RPA. These and other enzymatic properties of CeDna2 described in this paper may shed light on the observation that C.elegans is less stringently dependent on Dna2 for its viability than Saccharomyces cerevisiae. We propose that flaps generated by DNA polymerase δ-mediated displacement DNA synthesis are mostly short in C.elegans eukaryotes, and hence less dependent on Dna2 for viability.

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