Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1971 Oct;108(1):364–374. doi: 10.1128/jb.108.1.364-374.1971

Altered Deoxyribonucleic Acid Polymerase Activity in a Methyl Methanesulfonate-Sensitive Mutant of Bacillus subtilis

Kenneth B Gass a, Thomas C Hill a, Mehran Goulian a,1, Bernard S Strauss a, Nicholas R Cozzarelli a
PMCID: PMC247075  PMID: 4330738

Abstract

Of six deoxyribonucleic acid repair mutants of Bacillus subtilis assayed for deoxyribonucleic acid polymerase, only the methyl methanesulfonate-sensitive and ultraviolet light-sensitive mutant JB1-49(59) has impaired polymerase activity. Extracts prepared by sonic treatment or gentle lysis had about 10% of the wild-type activity with poly d(A–T), an alternating copolymer of deoxyadenylate and deoxythymidylate, used as template. The sensitivity to methyl methanesulfonate and ultraviolet light and the low level of polymerase activity transformed and reverted together, indicating that the two characteristics are a pleiotropic manifestation of a single mutation. Mixed extract and kinetic experiments mitigated against an altered nuclease activity as the enzymatic consequence of the mutation. Also, the mutant and wild type activities were stimulated equally by Escherichia coli exonuclease III. The residual activity in the mutant showed several differences from the wild-type activity: it purified differently, was more sensitive to sulfhydryl reagents, and displayed a different template specificity. We tentatively conclude that either the mutation in JB1-49(59) has introduced a qualitative as well as a quantitative change in the polymerase or the wild type contains two distinct polymerases, one of which is missing in the mutant.

Full text

PDF
364

Selected References

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

  1. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BOTT K., STRAUSS B. THE CARRIER STATE OF BACILLUS SUBTILIS INFECTED WITH THE TRANSDUCING BACTERIOPHAGE SP10. Virology. 1965 Feb;25:212–225. doi: 10.1016/0042-6822(65)90200-x. [DOI] [PubMed] [Google Scholar]
  3. Barbour S. D., Clark A. J. Biochemical and genetic studies of recombination proficiency in Escherichia coli. I. Enzymatic activity associated with recB+ and recC+ genes. Proc Natl Acad Sci U S A. 1970 Apr;65(4):955–961. doi: 10.1073/pnas.65.4.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyle J. M., Paterson M. C., Setlow R. B. Excision-repair properties of an Escherichia coli mutant deficient in DNA polymerase. Nature. 1970 May 23;226(5247):708–710. doi: 10.1038/226708a0. [DOI] [PubMed] [Google Scholar]
  5. Brutlag D., Atkinson M. R., Setlow P., Kornberg A. An active fragment of DNA polymerase produced by proteolytic cleavage. Biochem Biophys Res Commun. 1969 Dec 4;37(6):982–989. doi: 10.1016/0006-291x(69)90228-9. [DOI] [PubMed] [Google Scholar]
  6. Buttin G., Wright M. Enzymatic DNA degradation in E. coli: its relationship to synthetic processes at the chromosome level. Cold Spring Harb Symp Quant Biol. 1968;33:259–269. doi: 10.1101/sqb.1968.033.01.030. [DOI] [PubMed] [Google Scholar]
  7. De Lucia P., Cairns J. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature. 1969 Dec 20;224(5225):1164–1166. doi: 10.1038/2241164a0. [DOI] [PubMed] [Google Scholar]
  8. Dubnau D., Davidoff-Abelson R., Smith I. Transformation and transduction in Bacillus subtilis: evidence for separate modes of recombinant formation. J Mol Biol. 1969 Oct 28;45(2):155–179. doi: 10.1016/0022-2836(69)90097-7. [DOI] [PubMed] [Google Scholar]
  9. Gellert M., Bullock M. L. DNA ligase mutants of Escherichia coli. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1580–1587. doi: 10.1073/pnas.67.3.1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goldmark P. J., Linn S. An endonuclease activity from Escherichia coli absent from certain rec- strains. Proc Natl Acad Sci U S A. 1970 Sep;67(1):434–441. doi: 10.1073/pnas.67.1.434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Grossman L., Kaplan J. C., Kushner S. R., Mahler I. Enzymes involved in the early stages of repair of ultraviolet-irradiated DNA. Cold Spring Harb Symp Quant Biol. 1968;33:229–234. doi: 10.1101/sqb.1968.033.01.026. [DOI] [PubMed] [Google Scholar]
  13. HOTTA Y., BASSEL A. MOLECULAR SIZE AND CIRCULARITY OF DNA IN CELLS OF MAMMALS AND HIGHER PLANTS. Proc Natl Acad Sci U S A. 1965 Feb;53:356–362. doi: 10.1073/pnas.53.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Howard-Flanders P. DNA repair. Annu Rev Biochem. 1968;37:175–200. doi: 10.1146/annurev.bi.37.070168.001135. [DOI] [PubMed] [Google Scholar]
  15. Jovin T. M., Englund P. T., Bertsch L. L. Enzymatic synthesis of deoxyribonucleic acid. XXVI. Physical and chemical studies of a homogeneous deoxyribonucleic acid polymerase. J Biol Chem. 1969 Jun 10;244(11):2996–3008. [PubMed] [Google Scholar]
  16. Klein A., Niebch U. Host cell reactivation in strains of E. coli lacking DNA polymerase activity in vitro. Nat New Biol. 1971 Jan 20;229(3):82–84. doi: 10.1038/newbio229082a0. [DOI] [PubMed] [Google Scholar]
  17. Klenow H., Overgaard-Hansen K. Proteolytic cleavage of DNA polymerase from Escherichia Coli B into an exonuclease unit and a polymerase unit. FEBS Lett. 1970 Jan 15;6(1):25–27. doi: 10.1016/0014-5793(70)80032-1. [DOI] [PubMed] [Google Scholar]
  18. Knippers R. DNA polymerase II. Nature. 1970 Dec 12;228(5276):1050–1053. doi: 10.1038/2281050a0. [DOI] [PubMed] [Google Scholar]
  19. Kornberg T., Gefter M. L. DNA synthesis in cell-free extracts of a DNA polymerase-defective mutant. Biochem Biophys Res Commun. 1970 Sep 30;40(6):1348–1355. doi: 10.1016/0006-291x(70)90014-8. [DOI] [PubMed] [Google Scholar]
  20. Mahler I. Effect of mitomycin C on five excision-repair mutants of Bacillus subtilis. Biochem Biophys Res Commun. 1966 Oct 5;25(1):73–79. doi: 10.1016/0006-291x(66)90642-5. [DOI] [PubMed] [Google Scholar]
  21. Moses R. E., Richardson C. C. A new DNA polymerase activity of Escherichia coli. I. Purification and properties of the activity present in E. coli polA1. Biochem Biophys Res Commun. 1970 Dec 24;41(6):1557–1564. doi: 10.1016/0006-291x(70)90565-6. [DOI] [PubMed] [Google Scholar]
  22. OKAZAKI T., KORNBERG A. ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. XV. PURIFICATION AND PROPERTIES OF A POLYMERASE FROM BACILLUS SUBTILIS. J Biol Chem. 1964 Jan;239:259–268. [PubMed] [Google Scholar]
  23. OKUBO S., STRAUSS B., STODOLSKY M. THE POSSIBLE ROLE OF RECOMBINATION IN THE INFECTION OF COMPETENT BACILLUS SUBTILIS BY BACTERIOPHAGE DEOXYRIBONUCLEIC ACID. Virology. 1964 Dec;24:552–562. doi: 10.1016/0042-6822(64)90207-7. [DOI] [PubMed] [Google Scholar]
  24. Oishi M. An ATP-dependent deoxyribonuclease from Escherichia coli with a possible role in genetic recombination. Proc Natl Acad Sci U S A. 1969 Dec;64(4):1292–1299. doi: 10.1073/pnas.64.4.1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Okazaki R., Sugimoto K., Okazaki T., Imae Y., Sugino A. DNA chain growth: in vivo and in vitro synthesis in a DNA polymerase-negative mutant of E. coli. Nature. 1970 Oct 17;228(5268):223–226. doi: 10.1038/228223a0. [DOI] [PubMed] [Google Scholar]
  26. Okubo S., Romig W. R. Impaired transformability of Bacillus subtilis mutant sensitive to mitomycin C and ultraviolet radiation. J Mol Biol. 1966 Feb;15(2):440–454. doi: 10.1016/s0022-2836(66)80120-1. [DOI] [PubMed] [Google Scholar]
  27. Okubo S., Yanagida T. Isolation of a suppressor mutant in Bacillus subtilis. J Bacteriol. 1968 Mar;95(3):1187–1188. doi: 10.1128/jb.95.3.1187-1188.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pauling C., Hamm L. Properties of a temperature-sensitive radiation-sensitive mutant of Escherichia coli. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1495–1502. doi: 10.1073/pnas.60.4.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Prakash L., Strauss B. Repair of alkylation damage: stability of methyl groups in Bacillus subtilis treated with methyl methanesulfonate. J Bacteriol. 1970 Jun;102(3):760–766. doi: 10.1128/jb.102.3.760-766.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reiter H., Strauss B. Repair of damage induced by a monofunctional alkylating agent in a transformable, ultraviolet-sensitive strain of Bacillus subtilis. J Mol Biol. 1965 Nov;14(1):179–194. doi: 10.1016/s0022-2836(65)80239-x. [DOI] [PubMed] [Google Scholar]
  31. SCHACHMAN H. K., ADLER J., RADDING C. M., LEHMAN I. R., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. VII. Synthesis of a polymer of deoxyadenylate and deoxythymidylate. J Biol Chem. 1960 Nov;235:3242–3249. [PubMed] [Google Scholar]
  32. Searashi T., Strauss B. Glucose requirement for DNA breakdown resulting from treatment of Bacillus subtilis with ultraviolet radiation or methyl methanesulfonate. Mutat Res. 1967 May-Jun;4(3):372–375. doi: 10.1016/0027-5107(67)90032-2. [DOI] [PubMed] [Google Scholar]
  33. Searashi T., Strauss B. Relation of the repair of damage induced by a monofunctional alkylating agent to the repair of damage induced by ultraviolet light in Bacillus subtilis. Biochem Biophys Res Commun. 1965 Sep 22;20(6):680–687. doi: 10.1016/0006-291x(65)90069-0. [DOI] [PubMed] [Google Scholar]
  34. Strauss B. S. DNA repair mechanisms and their relation to mutation and recombination. Curr Top Microbiol Immunol. 1968;44:1–85. [PubMed] [Google Scholar]
  35. Strauss B., Reiter H., Searashi T. Recovery from ultraviolet- and alkylating-agent-induced damage in Bacillus subtilis. Radiat Res. 1966;(Suppl):201+–201+. [PubMed] [Google Scholar]
  36. Wilson G. A., Bott K. F. Nutritional factors influencing the development of competence in the Bacillus subtilis transformation system. J Bacteriol. 1968 Apr;95(4):1439–1449. doi: 10.1128/jb.95.4.1439-1449.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Witkin E. M. Ultraviolet mutagenesis in strains of E. coli deficient in DNA polymerase. Nat New Biol. 1971 Jan 20;229(3):81–82. doi: 10.1038/newbio229081a0. [DOI] [PubMed] [Google Scholar]
  38. Yasuda S., Sekiguchi M. Mechanism of repair of DNA in bacteriophage. II. Inability of ultraviolet-sensitive strains of bacteriophage in inducing an enzyme activity to excise pyrimidine dimers. J Mol Biol. 1970 Jan 28;47(2):243–255. doi: 10.1016/0022-2836(70)90343-8. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES