Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37°C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37 °C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469–8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the orc5-A strain's phenotype.

Origin recognition complex (ORC), a six-protein complex (Orc1p-Orc6p), may deeply involve in initiation of chromosomal DNA replication. However, since most temperature-sensitive orc mutants of Saccharomyces cerevisiae show the accumulation of cells with nearly 2C DNA content, the exact stage at which ORC acts is not fully understood. In this study, we constructed a heat-inducible degron mutant for each ORC subunit. As well as each targeted subunit, other subunits of ORC were also rapidly degraded under non-permissive conditions. In the orc5 degron mutant, incubation under the non-permissive conditions caused accumulation of cells with nearly 2C DNA content, and phosphorylation of Rad53p. When Orc5p (ORC) is depleted, this inhibits G1/S transition and formation of a pre-replicative complex (pre-RC). For pre-RC to form, and G1/S transition to proceed, Orc5p (ORC) must be present in late G1, rather than early G1, or G2/M. Block and release experiments revealed that Orc5p (ORC) is not necessary for S and G2/M phase progression. We therefore propose that ORC is necessary for the G1/S transition and pre-RC formation, and accumulation of cells with nearly 2C DNA content seen in various orc mutants is due to inefficient pre-RC formation, and/or induction of checkpoint systems.

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