Nucleic Acid Synthesis in Yeast

Wintersberger, Erhard

doi:10.1007/978-3-642-65908-9_3

Cited by 4 publications

(6 citation statements)

References 136 publications

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“…Specific activities of the enzyme preparations used in this work were between 6000 and 12000 units/mg protein for polymerase A and between 4000 and 6000 units/mg protein for polymerase B. As in earlier publications [1,2] one unit is defined as that amount of enzyme which catalyses the incorporation of 1 nmol of dTMP (16000 counts/min under our assay condition) into acid-insoluble product in 15 rnin at 35 "C with activated salmon sperm DNA as template-primer, (If this value is to be compared to total nucleotide polymerized/hour it has to be multiplied by 16.) It should be noted that DNA polymerase activity of highly purified preparations depends on the amounts of glycerol in the reaction mixture.…”

Section: Methodssupporting

confidence: 69%

“…DNA polymerases A and B were purified from diploid wild-type yeast as described previously [2] except that DNA-agarose was replaced by DNAcellulose [7] carrying denatured calf thymus DNA. This resulted in better purification of the enzymes.…”

Section: Methodsmentioning

confidence: 99%

“…It was observed earlier that yeast DNA polymerase B catalyzes a nucleolytic cleavage of DNA [13,2]. Since such an activity is not usually found in DNA polymerases of animal cells, it was of importance to verify with enzyme preparations of high purity that this nuclease activity is in fact associated with the polymerase B and is not due to an enzyme copurified with the polymerase.…”

Section: N a Polymerase B Contains An Associated 3'-exonuclease Actmentioning

confidence: 99%

“…C%rornriio,orripli~ of pur[fified DNA pollmrrosr A on DEAE-Sephndrx. 0.5 mg (1300 units) of DNA polymerase A fraction purified through the DNA-cellulose step[2] were adsorbed onto a column(1 x 6 cm) of DEAE-Sephadex A-25, equilibrated with 0.02 M Tris . HCI (pH 7.5),1 mM EDTA, 2 mM 2-mercaptoethanol and 10% glycerol.…”

mentioning

confidence: 99%

“…Polymerization of dTMI' by DNA polymerases A and B with diff'ercnt template-primers was measured as described [l 2]…”

mentioning

confidence: 99%

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Yeast DNA Polymerases: Antigenic Relationship, Use of RNA Primer and Associated Exonuclease Activity

Wintersberger¹

1978

European Journal of Biochemistry

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Highly purified preparations of DNA polymerases A and B from yeast were compared with respect to antigenic relationship, ability to use ribonucleotide primers and associated nuclease activity. The following results were obtained.1. Antiserum directed against DNA polymerase A inhibits this enzyme but does not interfere with the activity of DNA polymerase B or of mitochondrial DNA polymerase, nor does it precipitate the latter two enzymes.2. DNA polymerase A is capable of using oligo(ribouridy1ic acid) as a primer for the polymerization of dTMP. This reaction is not catalysed by polymerase B to any significant extent.3. Whereas DNA polymerase A is devoid of nuclease activity, DNA polymerase B catalyses an exonucleolytic release of mononucleotide units from the 3' end of polynucleotides. The results of several experiments suggest that this nuclease activity is associated with the DNA polymerase B molecule.In several earlier publications we reported on the isolation and partial characterization of the two nonmitochondrial DNA polymerases A and B present in the yeast Saccharomyces cerevisiue [l, 21. The two enzymes can be distinguished by a number of criteria. Having a molecular weight of 150000 -170000, both belong to the large-size class of DNA polymerases. Yeast DNA polymerase A has many properties in common with the DNA polymerase CI of animal cells [3]; an enzyme corresponding in size to the nuclear DNA polymerase p is lacking in yeast [4] and other lower eukaryotes [5].Here I present further characteristics which clearly distinguish yeast DNA polymerases A and B. The enzymes differ antigenically ; antibodies directed against and inhibiting DNA polymerase A have no effect on DNA polymerase B or on the mitochondrial DNA polymerase. Only DNA polymerase A can use oligoribonucleotide primers for the polymerization of thymidylate ; DNA polymerase B, but not polymerase, double-stranded copolymer of deoxyadenylic-thymidylic acid with alternating base sequence; poly(dA), poly(deoxyadeny1ic acid); poly(dA) . (dT),, , poly(deoxyadenylic acid) associated with deca(thymidy1ic acid) ( A : T = 1 : 1); (rU), , octa(ribouridy1ic acid).Definition. One AZ6(] unit is the quantity of polynuclcotidc contained in 1 ml of solution which has an absorbance of 1 at 260 nm when measured in a I-cm path-length cell.Enzyme. DNA polymerase (EC 2.7.7.7),

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Section: Methodssupporting

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Section: N a Polymerase B Contains An Associated 3'-exonuclease Actmentioning

confidence: 99%

mentioning

confidence: 99%

“…Polymerization of dTMI' by DNA polymerases A and B with diff'ercnt template-primers was measured as described [l 2]…”

mentioning

confidence: 99%

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Yeast DNA Polymerases: Antigenic Relationship, Use of RNA Primer and Associated Exonuclease Activity

Wintersberger¹

1978

European Journal of Biochemistry

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Chemistry and Biology of the Enterobacterial Common Antigen (ECA)

Mayer

Schmidt

1979

Current Topics in Microbiology and Immunology

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DNA-Dependent DNA Polymerase from Yeast Mitochondria. Dependence of Enzyme Activity on conditions of Cell Growth, and Properties of the Highly Purified Polymerase

Wintersberger

Blutsch

1976

Eur J Biochem

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The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on tlie carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide.Mitochondrial DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacrylamide gel electrophoresis revealed tlie following structure : a monomer of molecular weight around 60000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120000 which under low salt concentration (0.02 M Tris-HC1 buffer) formed higher aggregates. For optimal activity an M$+ ion concentration of 50 inM was found necessary, Mn ions did not promote activity at any concentration tested (0.5 -50 mM). Indeed, if added to M$+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2' ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50'%, decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the K, comentration. The enzyme preferred a gapped template primer, poly(dA) . (dT),,, over nicked DNA and was unable to use a polyribonucleotide template, poly(rt?) . (dT),, . In the purest preparations no exonuclease activity could be detected At least two DNA-synthesizing systems are operative in a eukaryotic cell: one in the nucleus, the other in the mitochondria. The recent progress concerning our knowledge of the mechanism of mitochondrial DNA synthesis [l -31 as well as the idea that the DNA-synthesizing apparatus of the organelles might perhaps be simpler and easier to investigate than that of the nucleus, prompted us to start isolating the components of the mitochondrial DNA-synthesizing inachinery.In an earlier publication [4] we had already described the properties of a partially purified preparation of DNA polymerase from yeast mitochondria

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Nucleic Acid Synthesis in Yeast

Cited by 4 publications

References 136 publications

Yeast DNA Polymerases: Antigenic Relationship, Use of RNA Primer and Associated Exonuclease Activity

Yeast DNA Polymerases: Antigenic Relationship, Use of RNA Primer and Associated Exonuclease Activity

Chemistry and Biology of the Enterobacterial Common Antigen (ECA)

DNA-Dependent DNA Polymerase from Yeast Mitochondria. Dependence of Enzyme Activity on conditions of Cell Growth, and Properties of the Highly Purified Polymerase

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