Saccharomyces cerevisiae cdc2 mutants arrest in the S‐phase of the cell cycle when grown at the non‐permissive temperature, implicating this gene product as essential for DNA synthesis. The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation. An open reading frame coding for a 1093 amino acid long protein with a calculated mol. wt of 124,518 was determined from the sequence. This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase. Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3′‐5′ exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta. These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.
DNA polymerase m from Saccharomyces cerevisiae is analogous to the mammalian DNA polymerase 8 by several criteria, including an increased synthetic activity on poly(dA)oligo(dT) (40:1 nucleotide ratio) in the presence ofcalf thymus proliferating-cell nuclear antigen (PCNA), or cyclin.This stimulation assay has been used to purify the yeast analog of PCNA/cyclin (yPCNA) to homogeneity. yPCNA is a trimer or tetramer (Mr 82,000) of identical subunits with a denatured Mr of 26,000. On a molar basis yPCNA and calf thymus PCNA/cyclin are equally active in stimulating DNA synthesis by DNA polymerase Im. About 10 times more yPCNA than calf thymus PCNA/cyclin is needed, however, to stimulate calf thymus DNA polymerase 8, and the degree of stimulation obtained at saturating levels of yPCNA is a factor of 2-3 less than with calf thymus PCNA/cyclin. Both stimulatory proteins exert their effect in an identical fashion, i.e., by increasing the processivity of the DNA polymerase. Yeast DNA polymerases I and H and calfthymus DNA polymerase a are not stimulated by yPCNA. Treatment of logarithmic-phase cells with hydroxyurea blocks them in the S phase and produces a 4-to 5-fold increase in yPCNA.Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is a cell cycle-regulated protein present at high levels in rapidly dividing cells (1-4). The intranuclear localization of PCNA/cyclin to regions of active DNA synthesis indicates that PCNA/cyclin plays a role in DNA replication (5). Recently, it has been shown that PCNA is required for in vitro simian virus 40 (SV40) DNA replication (6,7). In its absence, early replicative intermediates accumulate and leadingstrand DNA synthesis is impaired (7).The additional identification of PCNA/cyclin as an accessory factor of DNA polymerase 8 (8-10) suggests, in conjunction with the SV40 DNA replication data, a role for a complex of DNA polymerase 8 and PCNA/cyclin in processive leading-strand DNA synthesis. We have recently isolated from the yeast Saccharomyces cerevisiae a third nuclear DNA polymerase, which we call DNA polymerase III and which shows properties closely analogous to the mammalian DNA polymerase 8 (11). These include a proofreading 3' 5' exonuclease activity, sensitivity to the drug aphidicolin, and relative resistance to the nucleotide analog N2-[p-(n-butyl)phenylJdGTP (12). In addition, calf thymus PCNA/cyclin interacts with DNA polymerase III and promotes processive DNA synthesis by the enzyme (13). This finding prompted us to identify and purify a PCNA/cyclin-like activity in yeast by using stimulation of DNA polymerase III activity as an assay. The purification of this activity and its interaction with DNA polymerase III, as well as calf thymus DNA polymerase 8, is presented in this paper. Because the name cyclin implies that the expression of the gene and/or the activity of the protein is restricted to a part of the cell cycle, we prefer to use the more neutral term yPCNA (yeast proliferating-cell nuclear antigen) for the activity in yeast, in the absence...
The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication. It contains both DNA polymerase alpha (I) and delta (III). Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast. We have now cloned the gene for yeast PCNA (POL30). The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA. Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene. Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase. In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis.
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