ABSTRACT. The herpes simplex virus type 1 thymidine kinase gene TK complements the defect in five temperature- sensitive mutants and in vitro constructed ...
Proc. Nati. Acad. Sci. USA Vol. 81, pp. 5821-5825, September 1984 Genetics
Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK (Saccharomyces cerevisiae/DNA precursor mutant/cell-cycle gene product/DNA replication and repair)
ROBERT A. SCLAFANI AND WALTON L. FANGMAN Department of Genetics, SK-50, University of Washington, Seattle, WA 98195
Communicated by A. H. Doermann, May 31, 1984
The herpes simplex virus type 1 thymidine ABSTRACT kinase gene TK complements the defect in five temperaturesensitive mutants and in vitro constructed insertion and deletion mutants of the CDC8 gene of Saccharomyces cerevisiae. The herpes thymidine kinase enzyme acts as both a thymidine kinase and a thymidylate kinase (dTMP kinase). The latter activity is responsible for the cdc8 complementation since all thermosensitive cdc8 mutants are deficient in dTMP kinase activity at all temperatures. However, an intragenic revertant, cdc8-320, which was selected by demanding mitotic growth at the restrictive temperature, exhibits thermolabile dTMP kinase activity. We conclude that CDC8 is the structural gene for dTMP kinase, which catalyzes an essential step in DNA precursor biosynthesis. Previously, it had been shown that the DNA replication defect of cdc8 mutants could not be bypassed by the addition of deoxyribonucleoside triphosphates to permeabilized cells. This apparent discrepancy can be explained by hypothesizing a multiprotein yeast DNA replication complex containing the CDC8 protein.
The product of the CDC8 gene of Saccharomyces cerevisiae acts during the S phase of the yeast cell cycle (1, 2). Temperature-sensitive cdc8 mutants are defective in chromosomal, mitochondrial and 2-,um plasmid DNA replication (15) at the restrictive temperature. Most alleles of cdc8 also have a mutant phenotype at the permissive temperature; they exhibit an increase in spontaneous mitochondrial DNA mutability (6) and a decrease in ultraviolet light-induced mutagenesis (7). These properties of cdc8 mutants are consistent with a defect either in an essential replication protein, such as a DNA polymerase, or in a DNA precursor pathway. The latter possibility, however, appeared to be eliminated because thermolabile replication could not be bypassed in either permeabilized cells (8, 9) or a cell-free system (9-11) by providing the four deoxyribonucleoside triphosphates. In this communication, we provide evidence that CDC8 is, in fact, the structural gene for an enzyme of DNA precursor biosynthesis, thymidylate kinase (dTMP kinase). In vitro complementation assays have been used previously to isolate proteins that were thought to be the CDC8+ gene product (8, 11). One of these proteins is a 40-kilodalton DNA binding protein (11). The other is a 10- to 20-kilodalton protein (8). However, the sequence of the cloned CDC8+ gene encodes a 27-kilodalton protein (12, 13).
MATERIALS AND METHODS Bacterial Strains and Media. Escherichia coli strain JF1754 (hsdR metB leuB hisB lac gal) (14) was used for propagation of recombinant plasmids. Strains were grown in L broth with ampicillin (50 jig/ml) or tetracycline (10 ,.Lg/ml) and plasmids were isolated as described (15). M9 minimal media (16) was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
used for His' selection. Transformation was performed as described (17). Yeast Strains and Media. All yeast strains employed are S. cerevisiae and are listed in Table 1. All are nearly congenic with strain A364a (18) in that each was backcrossed more than eight times to A364a. Standard genetic methods were as described (19). Transformations were performed on whole cells (20) or spheroplasts (21). Yeast YEPD, YEPG, YM-1, and dropout media have been described (18, 22). Herpes TK+ activity in yeast was tested on inhibitor plates (14). Herpes TK- ASphI Construction. The 7.6-kilobase (kb) EcoRI fragment containing the HIS3' TK+ genes in plasmid pJM81 (14) was subcloned into the EcoRI site of plasmid YIP5 (23) by employing standard recombinant DNA methodology (15). The resulting plasmid, pRS244, contains two SphI sites, one in the pBR322 tetracycline gene (24) and the other located 385 base pairs into the herpes simplex virus type 1 (HSV-1) TK+ coding sequence (25). pRS244 was partially digested with DNA restriction endonuclease Sph I and the linearized 13.2-kb unit-length plasmid was purified by agarose gel electrophoresis (26). The DNA (0.4 ,ug) was digested with nuclease S1 (Miles Laboratories, 1 unit) at 23°C for 1 hr and then phenol extracted, ethanol precipitated, and blunt-end ligated with T4 DNA ligase (New England Biolabs). The ligation mixture was used to transform strain JF1754 and His' TetR colonies were selected. These colonies were screened by the rapid plasmid procedure (15) and clones harboring lasmids with only one Sph I site that is located in the Tet gene were chosen. Plasmid DNA from one such clone (TK- ASphI) was isolated and characterized. DNA restriction analysis revealed that, although the Sph I site in the herpes TK+ DNA was destroyed,
