Genetic and Molecular Analysis of the SOEl Gene : A tRNA : ' " Missense Suppressor of Yeast cdc 8 Mutations

The CDC8 gene of Saccharomyces cerevisiae encodes deoxythymidylate (dTMP) kinase and is required for nuclear and mitochondrial DNA replication in both the mitotic and meiotic cell cycles. All cdc8 temperature-sensitive mutants are partially defective in meiotic and mitochondrial functions at the permissive temperature. In a study of revertants of temperature-sensitive cdc8 mutants, the SOE201 and SOEl mutants were isolated. The SOE201 mutant is a disome of chromosome X to which the cdc8 gene maps. Using the chromosome X aneuploids to vary cdc8 gene dosage, we demonstrate that different levels of dTMP kinase activity are required for mitotic, meiotic or mitochondrial DNA replication. The SOEl mutant contains a dominant suppressor that suppresses five different cdc8 alleles but does not suppress a complete cdc8 deletion. The SOEl gene is located c1.5 cM from the CYH2 gene on chromosome VI1 and is adjacent to the TSM437-CYH2 region, with the gene order being SOEl-TSM437-CYH2. SOEl is an inefficient suppressor that can neither suppress the cdc8 hypomorphic phenotype nor restore dTMP kinase activity in vitro. SOEl is a single C to T mutation in the anticodon of a tRNA?" gene and thereby, produces a missense suppressor tRNA capable of recognizing AAA lysine codons. We propose that the resultant lysine to glutamate change stabilizes thermo-labile dTMP kinase molecules in the cell. C OORDINATION between the processes of DNA replication and biosynthesis of nucleotide precursors was first observed in prokaryotes. Proteins important for both processes may form multienzyme complexes or functional compartments (for a review see MATHEWS and ALLEN 1983). Several groups have also provided biochemical evidence for the existence of such multienzyme complexes in mammalian cells (REDDY and PARDEE 1980; WICKREMASINGHE, YAXLEY and HOFFBRAND 1983; for a review see MATHEWS and SLABAUCH 1986). We (SCLAFANI and FANGMAN 1984) and others (JONG, KUO and CAMPBELL 1984) have suggested that the yeast deoxythymidylate or dTMP kinase encoded by the CDC8 gene may be an integral part of such complexes. The analysis of mutations in genes important for nucleotide biosynthesis and for DNA replication can be a powerful tool for investigating this compartmentation hypothesis. For example, in bacteriophage T4, mutations in the gene for an enzyme important for precursor biosynthesis, dCMP-hydroxymethylase, can be suppressed by mutations in an enzyme important for DNA synthesis, DNA polymerase (CHAO, LEACH and KARAM 1977). Similarly, T 4 mutations that confer resistance to a School of Medicine, San Francisco, California 94 143. ' Present address: Department of Biochemistry, University of California of page charges. This article must therefore be hereby marked " du rtisement" The publication costs of this article were partly defrayed by the payment i n xcordance with 18 U.S.C. $1734 solely to indicate this fact. Genetics 124: 523-531 (March, 1990) folate analogue lie in the two genes that encode the subunits of DNA primase (MACDONALD and HALL 1984). We have isolated revertants of cdc8 temperature-sensitive mutants in an effort to identify other gene products that are important for either nucleotide biosynthesis or DNA replication (J.-Y. SU and R. A. SCLAFANI, unpublished results). We refer to these suppressors as SOE (suppressor of cdc eight) genes. However, one of these suppressors, SOEl, proved to be a novel, missense suppressor tRNA. Another suppressor, SOE201, was not a mutation but the result of increased cdc8 gene dosage by disomy. In this report, we describe a genetic and molecular characterization of the SOEl suppressor and an analysis of the effect of cdc8 gene dosage on mitotic, meiotic and mitochondrial chromosomal replication. MATERIALS AND METHODS Strains, plasmids and media: All yeast strains listed in Table 1 are Saccharomyces cereuisiae. The Escherichia coli bacterial strain used for transformation was DH5 recA end1 gyrA96 thi-1 hsdR17 supE44. Plasmids YRp7 SOC8-I (Kuo and CAMPBELL 1983) and pYe (CEN11)12 (FITZGERALDHAYES, BUHLER and COOPER 1982) were obtained from AMBROSE JONC and JOHN CARBON, respectively. Plasmid p5 1 1 1 A C E N l l was produced from plasmid p5 1 1-1 (Figures 1 and 2) by deletion of the 1.6-kb Sal1 C E N l I fragment by digestion with Sal 1 and ligation at a [DNA] of one fig/ ml to favor intramolecular ligation. DNA restriction fragments were subcloned into either plasmid pYe (CENl1)12 524 J.-Y. Su, L. Belmont and R. A. Sclafani