Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1976 Nov;3(11):3087–3099. doi: 10.1093/nar/3.11.3087

A simplified procedure for the analysis of DNA polymerase III levels in Bacillus subtilis strains.

G Ciarrocchi, A Fortunato, C Attolini, A Falaschi
PMCID: PMC343153  PMID: 826885

Abstract

A simple and reproducible procedure is described which allows the fast and almost quantitative removal of DNA polymerases I and II from DNA polymerase III, in crude extracts of polA+ strains of Bacillus subtilis. The procedure entails streptomycin sulfate and ammonium sulfate fractionations; subsequent analysis of the partially purified preparation by G-200 chromatography, DEAE cellulose chromatography and density gradient sedimentation, shows that the ammonium sulfate fraction contains less than 5% of the total activity as DNA polymerase I and less than 2% as DNA polymerase II. The purification procedure, up to the ammonium sulfate step, was utilized for the analysis of the level of DNA polymerase III in several B. subtilis mutants, with results comparable to those obtained from the corresponding polA- strains following more cumbersome purification procedures. The M.W. of the purified form is of 227.000, somewhat greater than the published values. The early fractions of the purification have revealed the existence of a form with a M.W. of 426.000; the nature of this form, which has been observed in several instances and which is very unstable and short-lived, is under investigation.

Full text

PDF
3087

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Clements J. E., D'Ambrosio J., Brown N. C. Inhibition of Bacillus subtilis deoxyribonucleic acid polymerase III by phenylhydrazinopyrimidines. Demonstration of a drug-induced deoxyribonucleic acid-enzyme complex. J Biol Chem. 1975 Jan 25;250(2):522–526. [PubMed] [Google Scholar]
  2. Cozzarelli N. R., Low R. L. Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication. Biochem Biophys Res Commun. 1973 Mar 5;51(1):151–157. doi: 10.1016/0006-291x(73)90521-4. [DOI] [PubMed] [Google Scholar]
  3. Ganesan A. T., Yehle C. O., Yu C. C. DNA replication in a polymerase I deficient mutant and the identification of DNA polymerases II and 3 in Bacillus subtilis. Biochem Biophys Res Commun. 1973 Jan 4;50(1):155–163. doi: 10.1016/0006-291x(73)91077-2. [DOI] [PubMed] [Google Scholar]
  4. Gass K. B., Cozzarelli N. R. Bacillus subtilis DNA polymerases. Methods Enzymol. 1974;29:27–38. doi: 10.1016/0076-6879(74)29006-2. [DOI] [PubMed] [Google Scholar]
  5. Gass K. B., Low R. L., Cozzarelli N. R. Inhibition of a DNA polymerase from Bacillus subtilis by hydroxyphenylazopyrimidines. Proc Natl Acad Sci U S A. 1973 Jan;70(1):103–107. doi: 10.1073/pnas.70.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gross J., Gross M. Genetic analysis of an E. coli strain with a mutation affecting DNA polymerase. Nature. 1969 Dec 20;224(5225):1166–1168. doi: 10.1038/2241166a0. [DOI] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Love E., D'Ambrosio D., Brown N. C. Mapping of the gene specifying DNA polymerase III of Bacillus subtilis. Mol Gen Genet. 1976 Mar 30;144(3):313–321. doi: 10.1007/BF00341730. [DOI] [PubMed] [Google Scholar]
  9. Low R. L., Rashbaum S. A., Cozzarelli N. R. Purification and characterization of DNA polymerase III from Bacillus subtilis. J Biol Chem. 1976 Mar 10;251(5):1311–1325. [PubMed] [Google Scholar]
  10. Monk M., Kinross J. Conditional lethality of recA and recB derivatives of a strain of Escherichia coli K-12 with a temperature-sensitive deoxyribonucleic acid polymerase I. J Bacteriol. 1972 Mar;109(3):971–978. doi: 10.1128/jb.109.3.971-978.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Noy G. C., Dalpra L., Pedrini A. M., Ciarrocchi G., Giulotto E., Nuzzo F., Falaschi A. Evidence for two waves of induction of DNA enzymes in stimulated human lymphocytes. Nucleic Acids Res. 1974 Sep;1(9):1183–1199. doi: 10.1093/nar/1.9.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Riva S., van Sluis C., Mastromei G., Attolini C., Mazza G., Polsinelli M., Falaschi A. A new mutant of Bacillus subtilis altered in the initiation of chromosome replication. Mol Gen Genet. 1975;137(3):185–202. doi: 10.1007/BF00333015. [DOI] [PubMed] [Google Scholar]
  13. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES