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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumATP bound to the origin recognition complex is important for preRC formationRichard D.Klemm and Stephen P.Bell* Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly. Initiation of DNA replication requires the precise and timely assembly of protein factors at each origin of replication. Recent work by a number of labs has identified a set of factors that localize to origins during the G1 phase of the cell cycle (1–5). In Saccharomyces cerevisiae, these higher-order complexes are nucleated by the origin recognition complex (ORC), which binds origin DNA in vitro and in vivo (1, 3, 6). ORC appears to be bound to origins throughout the cell cycle (4, 7) and is required for the stepwise recruitment of Cdc6p and a complex of six related proteins, Mcm2–7p, to the origin. This complex containing at least ORC, Cdc6p, and Mcm2–7p is known as the prereplicative complex (preRC; reviewed in ref. 8). Although originally identified in the yeast S. cerevisiae, subsequent studies have identified analogs of ORC and a similar preRC assembly process in multiple other eukaryotic species (reviewed in ref. 8). Ten of the 14 polypeptides known to be present in the S. cerevisiae preRC contain consensus nucleotide binding motifs within their sequences (9). In prokaryotic replication systems, ATP plays multiple roles in the initiation process (10, 11). Thus, it is likely that understanding the rol e of nucleotides in the eukaryotic initiation process will be important to determine the molecular details of this critical cellular event. Orc1p, Orc4p, Orc5p, Cdc6p, and Mcm2–7p all are members of a class of ATPases known as the AAA+ family (standing for ATPases associated with a variety of cellular activities; ref. 12). This family contains a region of sequence similarity that extends over 220–250 aa and includes the Walker A and B motifs common to many nucleotide binding proteins (reviewed in ref. 15). AAA+ members carry out diverse functions within the cell, including proteolysis, transcription, DNA replication, and recombination. A common functional theme for the role of ATP in these proteins is the regulation of the formation, rearrangement, and dissociation of macromolecular complexes (12). Typically, ATP binding stimulates the formation of the macromolecular complex and ATP hydrolysis stimulates disassembly (reviewed in ref. 13). We have previously demonstrated that the largest subunit of ORC, Orc1p, binds and hydrolyzes ATP (14). ATP binding to Orc1p is essential for ORC to specifically recognize and bind origins (6, 14). Furthermore, when ORC is bound to origin DNA, the ATPase activity of Orc1p is inhibited and ATP remains stably bound to Orc1p. We have hypothesized that this bound ATP could be hydrolyzed in a reaction coupled to a downstream step in replication, such as recruitment of other preRC components, initiation of replication, or inactivation of origins after initiation (14). To better understand the role of ATP hydrolysis in the function of ORC, we sought to generate Orc1p mutants that retain the ability to bind ATP, but lack hydrolysis activity. Mutation of the conserved Walker A motif within Orc1p leads to a loss of both ATP binding and hydrolysis activities. A second region common to many ATPases, the Walker B motif, is hypothesized to coordinate nucleotide hydrolysis activity rather than nucleotide binding. Consistent with this hypothesis, mutations within the B-motif of a number of ATPases have been identified that specifically affect ATP hydrolysis but not ATP binding (for examples, see refs. 16 and 17). Mutations that inhibit ATP hydrolysis but not ATP binding often possess a dominant lethal phenotype in vivo. For example, a mutant of the Escherichia coli initiator protein, DnaA, that can bind but not hydrolyze ATP causes overinitiation and lethality in a dominant manner (18). Similarly, a mutation in the Walker B motif of yeast Cdc6p leads to dominant lethality when overexpressed, whereas overexpression of wild-type Cdc6p or a mutant that is presumed to lack both ATP binding and hydrolysis activities does not cause lethality (19). Here, we describe the isolation and characterization of mutants within Orc1p that are dominant lethal when overexpressed and have hydrolysis-specific defects in vitro. We demonstrate that these mutants block replication by inhibiting preRC formation. Co-overexpressing Cdc6p suppresses the lethality of mutant ORC overexpression, suggesting that these mutant ORC complexes specifically titrate Cdc6p away from the origin. These findings support a model in which ATP binding to Orc1p stimulates higher order complex assembly and in which ATP hydrolysis by ORC stimulates preRC disassembly. Materials and MethodsPlasmids and Strains. Genotypes of yeast strains used in this study are listed in Table 1. Strains and plasmids were prepared by using standard laboratory methods (20). Plasmids to overexpress two of the ORC subunits were prepared by first subcloning the GAL1–10 promoter prepared by PCR from yeast genomic DNA This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: ORC, origin recognition complex; MCM, mini chromosome maintenance; ARS, autonomously replicating sequence; preRC, prereplicative complex; HA, hemagglutinin; ChIP, chromatin immunoprecipitation.
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