No abstract

Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have been identified by screening a collection of temperature-sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation. dna43-1 and dna52-1 cells undergo cell-cycle arrest at the restrictive temperature (37 degrees C), exhibiting a large-budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud and have failed to undergo DNA replication. These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase. DNA43 and DNA52 were cloned by their abilities to suppress the temperature-sensitive lethal phenotypes of dna43-1 and dna52-1 cells, respectively. DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34.

We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 11000 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictive temperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.

Bacteriophage OX174 cannot grow in a temperature-sensitive dnaC mutant of Escherichia coli C at the nonpermissive temperature. The inability to grow is the result of inhibition of virus DNA synthesis. Parental replicative form synthesis is not temperature sensitive. Single-stranded virus DNA continues to be synthesized for at least 45 min after shifting to the nonpermissive temperature late in infection. In contrast, the replication of the replicative form terminates within 5 min after shifting to the nonpermissive temperature.

The product of the Saccharomyces cerevisiae CDC8 gene is essential for normal cellular DNA replication; the determination of the structure of the gene and the identification of its product would facilitate the examination of its role in this process. We have cloned a 1,000-base-pair fragment of the S. cerevisiae genome carrying the functional gene. The nucleotide sequence includes one long open reading frame; it is flanked by sequences typical of other S. cerevisiae genes. This sequence predicts a polypeptide chain product of 216 amino acids with a molecular weight of 24,600. A polyadenylated RNA transcript of this sequence was identified by hybridization; in vitro translation of RNA samples enriched for this transcript produced a specific polypeptide chain of apparent molecular weight between 24,000 and 25,000. Thus the reading frame identified represents the authentic CDC8 gene, and the amino acid sequence of its product has been deduced. Our observations differ from two previous reports of the identification of the putative CDC8 protein based upon in vitro complementation assays.The effects of cdc8 mutations on the Saccharomyces cerevisiae cell division cycle have been extensively studied. The CDC8 gene product is required continuously during the S phase for normal DNA replication (8, 9). The mutant accumulates short replication bubbles at the restrictive temperature, suggesting that fork migration is defective (27). Short denaturation loops also appear under these conditions (11). Mitochondrial DNA synthesis (24, 34) and premeiotic DNA synthesis (28) are also defective in this mutant.The isolation of the functional CDC8 protein would allow a detailed study of its essential role in these DNA replication events. Several laboratories have developed cell-free DNA replication systems that appear to depend upon the CDC8 gene product (4, 10, 13). Attempts have been made to identify and isolate the CDC8 protein by using in vitro complementation assays based upon these cell-free systems. In one case the molecular weight of the putative CDC8 gene product was estimated to be 37,000 to 40,000, whereas in another case the preliminary estimate was 10,000 to 20,000 (1,14).If the CDC8 gene were cloned, one could examine its structure, identify its transcript, and characterize its protein product in a manner independent of these in vitro complementation assays. The cloning of a DNA segment carrying the functional CDC8 gene was recently reported (15). We report the results of studies here which have led to the determination of the nucleotide sequence of the CDC8 gene and the amino acid sequence of the authentic gene product. We discuss our findings relative to the earlier attempts to isolate the functional CDC8 protein. MATERIALS AND METHODSCells. Escherichia coli HB101 (F-thi leu pro hsdR hsdM recA endI) was obtained from J. D. Engel. S. cerevisiae strains A364A (a adel ade2 ural tyri his7 lys2 gall) and ts198 (cdc8-1 in same genetic background as A364A) were obtained from L. Hartwell. We note that the S. cerevisiae strain d...