Identification and reconstitution of the origin recognition complex from Schizosaccharomyces pombe

Abstract

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...

Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA

“…This idea suggests that the N-terminal region of spOrc4p, even when complexed with the other spOrc subunits, may be exposed. This notion is in keeping with the finding that the five-subunit complex, devoid of spOrc4p, is converted to the six-subunit complex upon addition of spOrc4p (19,20).…”

“…Previous studies showed that spORC binding to different origin sequences is mediated solely through the N-terminal AT-hook domains of Orc4p and that these interactions were not dependent on ATP (7,(18)(19)(20). A stable five-subunit spORC lacking Orc4p has been isolated as well as a six-subunit spORC containing an Orc4p derivative devoid of the N-terminal AT-hook domains (7,19,20). Such spORC derivatives do not bind to sp origin DNAs.…”

The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein

“…The presence of these multiple AT-hook motifs suggests that the binding of spORC to origin DNA is likely to be influenced by this domain. Although all the spORC subunits have been identified and the complex has been reconstituted from in vitro transcription and translation products of each subunit (17), the origin DNAbinding properties of spORC have not been examined as yet.…”

“…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.…”

Functional analysis of an Orc6 mutant in Drosophila

“…In S. cerevisiae, Orc6 is not important for DNA binding, but it is required for cell survival (10)(11)(12) and the maintenance of the pre-RC, specifically for recruitment of Cdt1 followed by MCM loading (13,14). Schizosaccharomyces pombe and metazoan Orc6 proteins (6,15,16) are more homologous, similar in size, and considerably smaller than the S. cerevisiae Orc6. In Drosophila, Orc6 is essential for ORC-dependent DNA binding and DNA replication (17,18).…”