Fig. 2. Biochemical characterization of an Orc1-d1p-containing complex. (A) ORC-d1 is defective in ATP hydrolysis. ATP hydrolysis was measured for ORC complexes containing wild-type Orc1p, an ATP binding-defective mutant Orc1p (ORC-1A) or Orc1-d1p. Hydrolysis was measured in the absence of DNA (filled bars) or in the presence of 7.5 pmol ARS1 DNA (open bars). One microgram of ORC (2.4 pmol) was used in each reaction. Rates are indicated as the amount of ATP hydrolyzed per pmol ORC. Each complex also contained a mutation in Orc5p that prevented ATP binding to this subunit (indicated by ORC-5A). (B) Kinetic analysis of ATP hydrolysis by the ORC complex. ATPase activity was measured for a complex containing wild-type ORC1 (Upper) or ORC-d1 (Lower) with a titration of ATP. Data were plotted as an Eadie-Hofstee plot. Calculated K_M and k_cat values are stated at the right. As in A, each complex contained the ORC-5A mutation. (C) The ORC-d1 complex can bind ARS1 DNA. Origin binding was assayed by using a DNase I protection assay for complexes containing wild-type Orc1p (lanes 2–4), ORC-1A (lanes 6–8), or Orc1-d1 (lanes 10–12). Lanes 2, 6, and 10 do not contain ATP. Lanes 3, 7, and 11 contain 100 µM ATP. Lanes 4, 8, and 12 contain 1.0 mM ATP. As in A, each complex also contained the ORC-5A mutation.

hydrolysis by ORC strongly suggests that the ORC-d1 complex will be almost exclusively in the ATP bound state yet be strongly defective in ATP hydrolysis. We believe that the loss of ATPase activity when ORC is not bound to DNA is the critical defect, because chromatin precipitation analysis of these mutant yeast strains indicates that the majority of overexpressed protein is found within the soluble fraction (data not shown).

orc1-d1 and orc1-d2 Support DNA Replication in Low Copy. In theory, the dominant lethal phenotype resulting from overexpressing the mutant ORC1 alleles could be caused by at least two different mechanisms. First, the mutant could compete with wild-type ORC for replication origin binding, but once bound be unable to perform an essential replication step, leading to lethality. Second, if the mutant ORC assumes a conformation that is preferentially recognized by an essential interacting factor, overexpression of the mutant ORC could titrate this factor away from the origin, leading to lethality. The first but not the second mechanism requires that the mutant ORC be unable to support DNA replication when the mutant allele is the only copy of ORC1 present in the cell.

To differentiate between these mechanisms, we tested whether the orc1-d1 and orc1-d2 mutants could complement a deletion of ORC1 when expression of the mutant genes was driven by the ORC1 promoter. Although the strains expressing orc1-d1 or orc1-d2 as the only copy of the ORC1 gene do have a growth defect (the generation time is 10–20% longer than wild-type controls), we found that both alleles complemented a deletion of ORC1 (Fig. 3A). We also used ChIP to test the ability of these mutants to bind origin DNA and assemble the preRC in vivo. ORC complexes containing Orc1-d1p or Orc1-d2p bound the origin efficiently (Fig. 3B). In addition, both mutant complexes assembled the preRC, as measured by MCM recruitment to the origin (Fig. 3C). These findings are consistent with the biochemical studies described above indicating that the ORC-d1 complex can bind ATP and DNA normally. We conclude that these mutants are capable of performing their essential function in replication and that the lethality induced by their overexpression is most likely due to titration of another essential replication factor away from origin bound ORC. Because neither wild-type nor ATP-binding-deficient ORC exhibits the lethal phenotype when overexpressed, we suggest that the inappropriately stabilized binding of non-origin bound ORC1-d1 or ORC1-d2 to ATP is responsible for titrating away the proposed replication factor.

Overexpression of Cdc6p Suppresses Orc1-d1 Lethality. We reasoned that, if a replication factor were being specifically titrated by the