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Introduction of an active enzyme into permeable cells of Escherichia coli

Acquisition of ultraviolet light resistance by uvr mutants on introduction of T4 endonuclease V

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Summary

Plasmolysed cells of Escherichia coli N212 (uvrA recA) acquired ultraviolet resistance when the cells were exposed to high concentrations of T4 endonuclease V. With increasing concentrations of T4 enzyme, survivals of plasmolysed cells after ultraviolet irradiation increased while colony-forming ability of unirradiated plasmolysed cells was not significantly affected by the enzyme treatment. Under appropriate conditions more than 200 fold increase in survivals was observed. When plasmolysed cells were treated with a pre-heated enzyme preparation or enzyme fractions derived from T4v 1(endonuclease V-deficient mutant)-infected cells, only little or no reactivation took place.

Permeabilization of cells prior to the enzyme treatment was essential for the effective reactivation. Treatment of intact cells with the T4 enzyme did not cause any reactivation. Cells treated with 20 mM EGTA or 50 mM CaCl2 in cold were reactivated to certain extents by the enzyme, but the extents of the reactivation were far less compared to those of plasmolysed cells.

Plasmolysed cells of strains carrying a mutation in one of uvrA, uvrB and uvrC genes were reactivated by introduction of T4 endonuclease V, as was the uvrA recA double mutant. UvrD mutants were also reactivated, but rather slightly. However, wild type strain as well as strains having a mutation in recA or polA gene were not reactivated. From these results it was suggested that T4 endonuclease V, taken up into permeable cells, can function in vivo to replace defective functions, which are controlled by the uvr genes. The conditions established in the present study may be used for introduction of other proteins into viable bacterial cells.

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References

  • Boyle, J.M., Paterson, M.C., Setlow, R.B.: Excision-repair properties of an Escherichia coli mutant deficient in DNA polymerase. Nature 226, 708–710 (1970)

    Google Scholar 

  • Braun, A., Grossman, L.: An endonuclease from Escherichia coli that acts preferentially on UV-irradiated DNA and is absent from the uvrA and uvrB mutants. Proc. Natl. Acad. Sci. USA. 71, 1838–1842 (1974)

    Google Scholar 

  • Buttin, G., Kornberg, A.: Enzymatic synthesis of deoxyribonucleic acid. XXI. Utilization of deoxyribonucleoside triphophates by Escherichia coli cells. J. Biol. Chem. 241, 5419–5427 (1966)

    Google Scholar 

  • Clark, A.J., Margulies, A.D.: Isolation and characterization of recombination-deficient mutants of Escherichia coli K12. Proc. Natl. Acad. Sci. USA. 53, 451–459 (1965)

    Google Scholar 

  • De Lucia, P., Cairns, J.: Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature 224, 1164–1166 (1969)

    Google Scholar 

  • Dorson, J.W., Moses, R.E.: DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli. J. Biol. Chem. 253, 665–670 (1978)

    Google Scholar 

  • Dürwald, H., Hoffmann-Berling, H.: Endonuclease I-deficient and ribonuclease I-deficient Escherichia coli mutants. J. Mol. Biol. 34, 331–346 (1968)

    Google Scholar 

  • Furusawa, M., Nishimura, T., Yamaizumi, M., Okada, Y.: Injection of foreign substances into single cells by cell fusion. Nature 249, 449–450 (1974)

    Google Scholar 

  • Harm, W.: Mutants of phage T4 with increased sensitivity to ultraviolet. Virology 19, 66–71 (1963)

    Google Scholar 

  • Harm, W.: Recovery of UV-inactivated E. coli cells by the ν-gene action of phage T4. Mutat. Res. 6, 175–179 (1968)

    Google Scholar 

  • Howard-Flanders, P., Boyce, R.P., Theriot, L.: Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics 53, 1119–1136 (1966)

    Google Scholar 

  • Howard-Flanders, P., Theirot, L., Stedeford, J.B.: Some properties of excision-defective recombination-deficients mutants of Escherichia coli K-12. J. Bact. 97, 1134–1141 (1969)

    Google Scholar 

  • Kato, T.: Excision repair characteristics of recB - res-and uvrC -strains of Escherichia coli. J. Bact. 112, 1237–1246 (1972)

    Google Scholar 

  • Lederberg, J., Clair, J. ST.: Protoplasts and L-type growth of Escherichia coli. J. Bact. 75, 143–160 (1958)

    Google Scholar 

  • Leive, L.: A nonspecific increase in permeability in Escherichia coli produced by EDTA. Proc. Natl. Acad. Sci. USA. 53, 745–750 (1965)

    Google Scholar 

  • Low, B.: Formation of merodiploids in mating with a class of Rec -recipient strains of Escherichia coli K12. Proc. Natl. Acad. Sci. USA. 60, 160–167 (1968)

    Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951)

    Google Scholar 

  • Loyter, A., Zakai, N., Kulka, R.G.: “Ultramicroinjection” of macromolecules or small particles into animal cells: A new technique based on virus-induced cell fusion. J. Cell Biol. 66, 292–304 (1975)

    Google Scholar 

  • Mandel, M., Higa, A.: Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159–162 (1970)

    Google Scholar 

  • Miura, A., Tomizawa, J.: Studies on radiation-sensitive mutants of E. coli III. Participation of the Rec system in induction of mutation by ultraviolet irradiation. Mol. Gen. Genet. 103, 1–10 (1968)

    Google Scholar 

  • Moses, R.E.: Replicative deoxyribonucleic acid synthesis in a system diffusible for macromolecules. J. Biol. Chem. 247, 6031–6038 (1972)

    Google Scholar 

  • Moses, R.E., Richardson, C.C.: Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc. Natl. Acad. Sci. USA. 67, 674–681 (1970)

    Google Scholar 

  • Ogawa, H.: Genetic locations of uvrD and pol genes of E. coli. Mol. Gen. Genet. 108, 378–381 (1970)

    Google Scholar 

  • Ogawa, H., Shimada, K., Tomizawa, J.: Studies on radiation-sensitive mutants of E. coli I. Mutants defective in the repair synthesis. Mol. Gen. Genet. 101, 227–244 (1968)

    Google Scholar 

  • Otsuji, N., Higashi, T., Kawamata, J.: Genetic and physiological analysis of mitomycin C-sensitive mutants of Escherichia coli K12. Biken J. 15, 49–59 (1972)

    Google Scholar 

  • Papahadjopoulos, D., Poste, G., Mayhew, E.: Cellular uptake of cyclic AMP captured within phospholipid vesicles and effect on cell-growth behaviour. Biochim. Biophys. Acta. 363, 404–418 (1974)

    Google Scholar 

  • Sato, K., Sekiguchi, M.: Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: The structural gene for T4 endonuclease V. J. Mol. Biol. 102, 15–26 (1976)

    Google Scholar 

  • Seeberg, E.: Reconstitution of an Escherichia coli repair endonuclease activity from the separated uvrA +and uvrB +/uvrC+gene products. Proc. Natl. Acad. Sci. USA. 75, 2569–2573 (1978)

    Google Scholar 

  • Seeberg, E., Johansen, I.: Incisions in ultraviolet irradiated circular bacteriophage λ DNA molecules in excision proficient and deficient lysogens of E. coli. Mol. Gen. Genet. 123, 173–184 (1973)

    Google Scholar 

  • Seeberg, E., Nissen-Meyer, J., Strike, P.: Incision of ultraviolet-irradiated DNA by extracts of E. coli requires three different gene products. Nature 263, 524–526 (1976)

    Google Scholar 

  • Seeberg, E., Strike, P.: Excision repair of ultraviolet-irradiated deoxyribonucleic acid in plasmolyzed cells of Escherichia coli. J. Bacteriol. 125, 787–795 (1976)

    Google Scholar 

  • Sekiguchi, M., Shimizu, K., Sato, K., Yasuda, S., Ohshima, S.: Enzymic mechanism of excision-repair in T4-infected cells. In: Molecular mechanisms for repair of DNA (ed. by P.C. Hanawalt and R.B. Setlow), Part A, pp. 135–142. New York: Plenum Press 1975

    Google Scholar 

  • Shimada, K., Ogawa, H., Tomizawa, J.: Studies on radiation-sensitive mutants of E. coli II. Breakage and repair of ultraviolet irradiated intracellular DNA of phage lambda. Mol. Gen. Genet. 101, 245–256 (1968)

    Google Scholar 

  • Shimizu, K., Sekiguchi, M.: 5′→3′ Exonucleases of bacteriophage T4. J. Biol. Chem. 251, 2613–2619 (1976)

    Google Scholar 

  • Shimizu, K., Sekiguchi, M.: Insertion of a functional repair enzyme into viable cells of Escherichia coli. Proceedings of the 1977 molecular biology meeting of Japan, pp. 93–95 (1977)

  • Smith, K.C., Meun, D.H.C.: Repair of radiation-induced damage in Escherichia coli I. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation. J. Mol. Biol. 51, 459–472 (1970)

    Google Scholar 

  • Stafford, M.E., Reddy, G.P.V., Mathews, C.K.: Further studies on bacteriophage T4 DNA synthesis in sucrose-plasmolyzed cells. J. Virol. 23, 53–60 (1977)

    Google Scholar 

  • Taketo, A., Yasuda, S., Sekiguchi, M.: Initial step of excision repair in Escherichia coli: Replacement of defective function of uvr mutants by T4 endonuclease. V. J. Mol. Biol. 70, 1–14 (1972)

    Google Scholar 

  • Tanaka, K., Hayakawa, H., Sekiguchi, M., Okada, Y.: Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo. Proc. Natl. Acad. Sci. USA. 74, 2958–2962 (1977)

    Google Scholar 

  • Tanaka, K., Sekiguchi, M., Okada, Y.: Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus). Proc. Natl. Acad. Sci. USA. 72, 4071–4075 (1975)

    Google Scholar 

  • Vosberg, H-P., Hoffmann-Berling, H.: DNA synthesis in nucleotide-permeable Escherichia coli cells I. Preparation and properties of ether-treated cells. J. Mol. Biol. 58, 739–753 (1971)

    Google Scholar 

  • Wickner, R.B., Hurwitz, J.: DNA replication in Escherichia coli made permeable by treatment with high sucrose. Biochem. Biophys. Res. Commun. 47, 202–211 (1972)

    Google Scholar 

  • Wovcha, M.G., Tomich, P.K., Chiu, C., Greenberg, G.R.: Direct participation of dCMP hydroxymethylase in synthesis of bacteriophage T4 DNA. Proc. Natl. Acad. Sci. USA. 70, 2196–2200 (1973)

    Google Scholar 

  • Yamamoto, Y.: Studies on DNA degradation after UV-irradiation in uvrD -mutant of Escherichia coli. Proceedings of the 1976 molecular biology meeting of Japan, pp. 136–137 (1976)

  • 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. 47, 243–255 (1970a)

    Google Scholar 

  • Yasuda, S., Sekiguchi, M.: T4 endonuclease involved in repair of DNA. Proc. Natl. Acad. Sci. USA. 67, 1839–1845 (1970b)

    Google Scholar 

  • Yasuda, S., Sekiguchi, M.: Further purification and characterization of T4 endonuclease. V. Biochim. Biophys. Acta. 442, 197–207 (1976)

    Google Scholar 

  • Zinder, N.D., Arndt, W.F.: Production of protoplasts of Escherichia coli by lysozyme treatment. Proc. Natl. Acad. Sci. USA. 42, 586–590 (1956)

    Google Scholar 

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  1. Department of Biology, Faculty of Science, Kyushu University 33, 812, Fukuoka, Japan

    Kenji Shimizu & Mutsuo Sekiguchi

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  1. Kenji Shimizu
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  2. Mutsuo Sekiguchi
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Communicated by T. Yura

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Shimizu, K., Sekiguchi, M. Introduction of an active enzyme into permeable cells of Escherichia coli . Molec. Gen. Genet. 168, 37–47 (1979). https://doi.org/10.1007/BF00267931

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