Synchronized Yeast Cells

Eckstein, H.‐H.; Paduch, V.; Hilz, Helmuth

doi:10.1111/j.1432-1033.1967.tb19520.x

Cited by 35 publications

(7 citation statements)

References 25 publications

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“…A similar fluctuation of DNA polymerase activity in cell homogenates has also been observed in synchronized yeast cells in which DNA polymerase activity increases about 2-fold during initiation of DNA synthesis [34,22]. A difference in the specific polymerase activity is also apparent in extracts prepared from growing or resting cells; in a growing cell population in which approximately 30% of the cells are in S-phase, DNA polymerase activity is about 1.4 times higher than in resting cells.…”

Section: Dna-polymerase a C Tivity In Growing And Resting Yeast Cellssupporting

confidence: 60%

Deoxyribonucleic Acid Polymerases from Yeast

Wintersberger

1974

European Journal of Biochemistry

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Yeast DNA polymerases A and B were purified 5000-10000-fold by chromatography on DEAEcellulose, phosphocellulose, DNA-agarose and DEAE-Sephadex. In acrylamide gel electrophoresis in the presence of sodium dodecylsulfate, both enzymes give rise to three main bands. The enzymes are equally susceptible to inhibition by the -SH reagents N-ethylmaleimide and p-chloromercuribenzoate but differ in their sensitivity to cytosine-arabinoside triphosphate, polymerase A being considerably more sensitive to this nucleotide analog. Whereas DNA polymerase A prefers nicked DNA and poly[d(A-T)] as template-primer, polymerase B is most active with poly(dA) . (dT),,.K , values for deoxyribonucleoside triphosphates were determined as 3.7 -3.9 pM for enzyme A and 1.8 -2.4 pM for enzyme B. During active growth of cells polymerase activity increases about 1 .4-fold over the amount found in resting cells, which is mainly if not exclusively due to an increased level of DNA polymerase A. This together with other properties suggests a role of this enzyme in DNA replication.Eukaryotes like prokaryotes contain several DNA polymerases [l -111 the specific role of which is still incompletely understood. While the use of mutants has helped to clarify some principal questions concerning DNA synthesis in bacteria [12], in most higher cells similar techniques cannot be applied. One simple eukaryote which is amenable to genetic analysis is yeast and we have therefore initiated studies on DNA replication in this organism. Our previous experiments have shown that besides the mitochondria1 DNA polymerase [13], two enzymes, DNA polymerases A and B, can be isolated from yeast homogenates. These enzymes were partially purified and characterized [5]. With regard to their size they correspond to the 6 -8-S type of DNA polymerases, which are found in the cytoplasm of animal cells. En:jine. DNA polymerase or deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase (EC 2.7.7.7). ~~In this paper I want to summarize results of continued studies on yeast DNA polymerases A and B which were primarily concerned with the further purification of the enzymes and attempts to clarify whether the two polymerases are distinct enzymes, with different properties and functions. MATERIALS AND METHODSThe deoxyribonucleoside triphosphates, poly Poly(rA) . (dT)~o and ~0 1~-(dA) . poly(dT), were from Boehringer Mannheim GmbH (Mannheim, Germany), tritiated deoxyribonucleoside triphosphates from the Radiochemical Centre (Amersham). Poly(dA) . (dT),, was purchased from P. L. Laboratories, cytosine-arabinoside triphosphate from Sigma Chemical Co. Salmon sperm DNA (Sigma, type 111) was activated by treatment with pancreatic deoxyribonuclease as described earlier [5]. DEAE-cellulose (DE 52) and phosphocellulose (P-11) were from Whatman. The ion-exchange resins were precycled with 0.5 M NaOH and 0.5 M HCI and then equilibrated with the buffer used for chromatogEur. J. Biochem. 50 (1974)

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Section: Dna-polymerase a C Tivity In Growing And Resting Yeast Cellssupporting

confidence: 60%

Deoxyribonucleic Acid Polymerases from Yeast

Wintersberger

1974

European Journal of Biochemistry

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“…Unless otherwise indicated, the standard reaction mixture contained, in a final volume of 0.125 ml: 0.05 M Tris-C1, pH 7.5, I0 mM MgCl,, 1 mM EDTA, 2 mM B-mercaptoethanol, 50p.M each of dGTP, dATP, dCTP, and dTTP, 0.25 p.C [3H]dTTP, 13 pg of "activated" salmon sperm DNA and enzyme. 125 pg of phosphoenol pyruvate (trisodium salt) and 10 pg of pyruvate b a s e were added to each assay for the activity determinations of enzyme preparations prior to the DEAE-cellulose chromatography step. After 15 min incubation a t 35", the reaction was stopped by chilling and loo$ of the mixture were applied to discs For crude enzyme solutions this assay is linear up to an amount of about 100 pg of input protein; for more purified preparations, after the DEAEcellulose chromatography step, linearity is observed only up to 20 pg of input protein.…”

Section: Determination Of D N a Polymerase Activitymentioning

confidence: 99%

Studies on Deoxyribonucleic Acid Polymerases from Yeast

Wintersberger¹,

Wintersberger

1970

European Journal of Biochemistry

105

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A mitochondria-free cell extract from Xaccharomyces cerevisiae was used as source of yeast DNA polymerase for the partial purification and characterization of the enzyme. Upon DEAEcellulose chromatography, the total DNA polymerase activity separates into a major fraction, DNA polymerase A, and a minor fraction, DNA polymerase B. The final purification achieved was 150-to 200-fold for DNA polymerase A and 60-to 80-fold for DNA polymerase B. The two polymerases A and B differ from each other in some properties. Both purified enzyme preparations contain only traces of nucleases and very little, if any, terminal transferase. Activity is totally dependent on the presence of DNA, all four deoxyribonucleoside triphosphates and Mg++. With "activated" DNA or with poly d(A-T) as template the DNA polymerases A and B exhibit a n optimal activity. Native DNA preparations promote DNA synthesis to a low degree only, while denatured DNA9 are even less active as templates. The enzymes have a molecular weight around 150 000 ; however, larger aggregates have been observed under certain conditions. DNA polymerases have been purified and characterized from a variety of bacterial and animal sources. These enzymes synthesize DNA from monomeric units, the deoxyribonucleoside triphosphates, the base sequence being determined by a DNA template. The recent extensive studies in Kornberg's laboratory on a homogeneous preparation of Escherichia coli DNA polymerase have clarified much of the mechanism of action of this enzyme [l-61. I n the last few years it has become increasingly clear, however, that the process of DNA replication in the living cell cannot be mediated solely by DNA polymerase but that this enzyme is only part of a more complicated replication machinery. (An extensive discussion of all aspects of DNA synthesis in microorganisms can be found elsewhere [6 a] .)Eukaryotic cells present a further interesting feature of cellular DNA synthesis because these cells contain partially autonomous particles, mitochondria and chloroplasts, which have their own DNA replicating machinery [7].I n the course of our own studies on mitochondria1 DNA polymerase from yeast [S,

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“…Previously, Eckstein et al (12) had reported that intracellular activity of DNA polymerase in the synchronously cultured cells of S. cerevisiae alters in an oscillating manner with the maximal activity at just before initiation of DNA replication in the cell division cycle. In this report, we found that the intranuclear specific-activity of the polymerase characteristically increases in dependent fashion on prograssion of the cell division cycle, from middle to late Gl phase, with little alteration in cellular activity of the enzyme (specific activity) by using cdc-mutants, cdc 25, cdc 28 cdc 4 and cdc 7.…”

Section: Discussionmentioning

confidence: 99%

Characteristic alteration in the nuclear DNA polymerase activity during the cell division cycle ofSaccharomyces cerevisiae

Tsuchiya¹,

Kimura²,

Miyakawa³

et al. 1984

Nucl Acids Res

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Specific activity of the intranuclear DNA polymerase in cdc-mutant cells of Saccharomyces cerevisiae was found to be characteristically changed by arrest in their specific stage of cell division cycle without a notable alteration in the total cellular activity. The activities were low in the nuclei of cdc 25, cdc 28 and cdc 4, which were arrested in early to mid G1 phase by temperature shift-up, and in the nuclei of wild-type cells (A364A), which were arrested in early G1 phase by alpha-factor treatment, while high level of the activity was found in the nuclei of cdc 7 and cdc 8, which were arrested at late G1 and S phase, respectively. Activity-gel analysis of DNA polymerase in the nuclear extracts revealed the presence of two active peptides (120K and 72K), and the characteristic decrease in both active peptides was induced by arrest in early to mid G1 phase. Consequently, it is strongly suggested that intranuclear DNA polymerase activity alters in a dependent fashion on progression of cell division cycle. Subunit analysis indicated that the purified DNA polymerase I is constructed from two subunit peptides of 120K and 62K, and the large subunit possesses catalytic activity.

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Synchronized Yeast Cells

Cited by 35 publications

References 25 publications

Deoxyribonucleic Acid Polymerases from Yeast

Deoxyribonucleic Acid Polymerases from Yeast

Studies on Deoxyribonucleic Acid Polymerases from Yeast

Characteristic alteration in the nuclear DNA polymerase activity during the cell division cycle ofSaccharomyces cerevisiae

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