Activity of yeast extracts in cell - free stimulation of DNA replication ( DNA synthesis in vitro / growth control / cell cycle / cdc mutants )

Extracts of the cytoplasm of disrupted spheroplasts of Saccharomyces cerevisiae (bakers' yeast) stimulated DNA synthesis in a cell-free system consisting of nuclei from spleen cells of the frog Xenopus laevis. The stimulation required Mg++, ATP, and the deoxynucleoside triphosphates, was saturated by an excess of nuclei or extract, and had kinetics resembling those obtained previously with extracts from mammalian and avian cells. After addition of the yeast extract, replication "eyes" were formed in the DNA from the nucleochromatin of the frog, suggesting that the extract stimulated the initiation of DNA replication. The activity was susceptible to heat, was nondialyzable, and was abrogated by tryptic digestion. Temperature-sensitive mutants of the cell division cycle (cdc mutants 4, 7,8, and 28) grown at permissive temperature (23°) yielded extracts that were capable of stimulating DNA replication. When the cells were incubated for one generation at the nonpermissive temperature (360), their extracts showed very low or no activity. All of these mutants are deficient in events of the dependent pathway leading to initiation of DNA synthesis in the yeast cell cycle. A ts mutant, cdclO, deficient in the separate pathway for cytokinesis, showed little or no loss of activity at the nonpermissive temperature. These data indicate that the "initiation" activity, as assayed in vitro, is subject to control in the yeast cell cycle, and -its appearance may be one of the terminal events in the pathway leading to DNA synthesis. The finding that extracts from yeast cells can stimulate DNA synthesis in nucleochromatin from frog cells, and the fact that the cdc mutants 4, 7,8, and 28 describe a dependent pathway terminating in development of "initiation" activity, are in accord with the hypothesis that the function of proteins in the dependent pathways of the cell cycle is conserved during evolution. An understanding of the biochemistry of the cell cycle in eukaryotes requires a molecular analysis of the nature of the initial signals and the subsequent controls of a highly ordered sequence of events resulting in DNA synthesis and cell division. It is particularly important to understand the regulation of the initial events commiting a cell to the entry from a resting stage to the Gi phase of the cycle. This requires: (i) a means of establishing the order of biochemical events that are essential for the initiation of DNA synthesis; and (ii) assays that will allow isolation of the molecules responsible. A particularly significant step towards realizing the first of these objectives has been taken by Hartwell and his colleagues (1). Using a series of temperature-sensitive yeast mutants, they have provided an explanation for the orderly temporal sequence of events in terms of the dependence of each event on prior events in the cycle. The second objective, isolation of the molecules involved in each step, requires the development of suitable biochemical approaches. We have recently described a cell-free assay for the onset of DNA synthesis (2). Using this assay, we have shown that cytoplasmic factors from proliferating but not from resting cells of various kinds can induce the onset of DNA replication in isolated nuclei of cells from adult frog spleens and livers. Abbreviation: cdc, temperature-sensitive mutants of the yeast cell division cycle (1). 3933 Moreover, we provided evidence that the appearance of the active factors is under control and can be influenced by density-dependent inhibition, by mitogens, and by nonpermissive conditions in cells transformed by temperature-sensitive viral mutants. In the present paper, we describe the application of the "initiation" assay to an analysis of the factors of wild-type and selected cell division cycle (cdcf mutants of yeast (1, 3). This may be useful in the isolation and characterization of the proteins involved in initiating DNA replication. MATERIALS AND METHODS [methyl-3H]dTTP (50 Ci/mmol) was from Schwarz/Mann, nucleotides were from P-L Biochemicals, and glusulase from Endo Laboratories (Garden City, N.Y.) Yeast Strains and Cultures. Saccharomyces cerevisiae (bakers' yeast) was from Fleischmann (New York, N.Y.) and was used in the experiments described in Figs. 1 and 2, and in Tables 1 and 2. The temperature-sensitive cell division cycle mutants of S. cerevisiae, cdc4-1, cdc7-1, cdc8-1, cdclo-1, and cdc28-1, as well as the parent strain A364A were from the Yeast Genetic Stock Center (University of California, Berkeley, Calif.). Yeast cells (Fleischmann's Yeast) were grown in 2% peptone-1% yeast extract-2% glucose (Difco) on a shaker at 300. The other strains (A364A and the cdc mutants) were grown in the same medium at 23', the permissive temperature. Preparation of Cell Extracts and Nuclei. Exponential yeast cultures were harvested and the cells converted to spheroplasts according to the method of Kuo and Lampen (4). The spheroplasts (3 X 109 per ml) were suspended in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (Hepes) (pH 85)-5 mM KCI-0.5 mM MgCl2-0. 1 mM (ethylene dinitrilo)tetraacetate (EDTA)-0.5 mM dithiothreitol, and extracts were prepared as described (2). This procedure disrupted the spheroplasts, leaving many intact but swollen nuclei. The extracts consisted mainly of cytoplasmic material, although the presence of some nuclear material cannot be excluded. Nuclei were prepared from frog spleen cells (Xenopus laevis, NASCO, Fort Atkinson, Wisc.) as before (2). These cells are composed mainly of resting cells that do not replicate their DNA (2). Other Methods. The assay for chromosomal DNA synthesis has been described (2) and was performed at 300. Samples were prepared for electron microscopy (2) and the DNA was visualized by the aqueous spreading technique of Davis et al. (5) with uranyl acetate staining and no shadowing. Duplex circular DNA of bacteriophage fl (a gift of Dr. Peter Model of The Rockefeller University) was used as a 2-Am length standard.