Abstract
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 degrees 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.
Highlights
The initiation of chromosomal DNA replication is tightly regulated to achieve genome replication just once per cell cycle
We examined the role of ATP binding to Orc5p in chromosomal DNA replication in cells by replacing the wild-type ORC5 gene on S. cerevisiae chromosome with the orc5-A gene to construct the orc5-A strain
We showed that the YY411 strain expressing ORC5-A (ORC with Orc5pK43E, a mutation in the ATP-binding domain) showed temperature-sensitive growth, and we found that the amount of origin recognition complex (ORC) is decreased at a nonpermissive temperature (37 °C)
Summary
Plasmids, and Medium—The S. cerevisiae strains are listed in Table I [20, 21]. The ORC5 gene was amplified by PCR from chromosomal DNA of the W303-1A strain and inserted into pRS416 [22], a low copy number plasmid containing the URA3 gene, to create pRS416-ORC5, which was used for the plasmid shuffling experiments. The orc5-A gene (orc5K43E) was introduced into pRS406 (another plasmid containing the URA3 gene) [22] to create pRS406-orc5-A This plasmid was transformed into W303-1A, and the transformed cells were selected on SC agar plates lacking uracil. To overexpress each ORC subunit and Cdc6p in cells, the genes for these proteins were amplified by PCR from chromosomal DNA of the.
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