Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1984 Jan;157(1):21–27. doi: 10.1128/jb.157.1.21-27.1984

Physical and biochemical analysis of the cloned recB and recC genes of Escherichia coli K-12.

C C Dykstra, D Prasher, S R Kushner
PMCID: PMC215123  PMID: 6317651

Abstract

A 19-kilobase BamHI fragment encoding the recB (exonuclease V), recC (exonuclease V), ptr (protease III), thyA, and argA genes of Escherichia coli K-12 was cloned into a multicopy plasmid (pCDK3). In E. coli maxicells, the plasmid specified the synthesis of seven polypeptides of 140,000 (recC), 128,000 (recB), 110,000 (ptr), 53,000 (argA), 50,000, 33,000 (thyA), and 22,000 Mr, as well as beta-lactamase and chloramphenicol acetyltransferase. From analysis of subclones and Tn1000 insertions, it appears that the 110,000- and 50,000-Mr proteins originated from the ptr DNA coding sequence which is located between the recB and recC genes. Although recC, ptr, and recB were physically closely linked and transcribed in the same direction, they do not appear to constitute an operon. Cells carrying pCDK3 contained a 30- to 50-fold increase in exonuclease V activity, without affecting cell viability.

Full text

PDF
21

Images in this article

24Image
on p.24
24Image
on p.24

Selected References

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

  1. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev. 1983 Jun;47(2):180–230. doi: 10.1128/mr.47.2.180-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Belfort M., Maley G. F., Maley F. Characterization of the Escherichia coli thyA gene and its amplified thymidylate synthetase product. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1858–1861. doi: 10.1073/pnas.80.7.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E., Furlong L., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. doi: 10.1126/science.847462. [DOI] [PubMed] [Google Scholar]
  5. Brandsma J. A., Stoorvogel J., van Sluis C. A., van de Putte P. Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12. Gene. 1982 Apr;18(1):77–85. doi: 10.1016/0378-1119(82)90058-0. [DOI] [PubMed] [Google Scholar]
  6. CLARK A. J., MARGULIES A. D. ISOLATION AND CHARACTERIZATION OF RECOMBINATION-DEFICIENT MUTANTS OF ESCHERICHIA COLI K12. Proc Natl Acad Sci U S A. 1965 Feb;53:451–459. doi: 10.1073/pnas.53.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cheng Y. S., Zipser D., Cheng C. Y., Rolseth S. J. Isolation and characterization of mutations in the structural gene for protease III (ptr). J Bacteriol. 1979 Oct;140(1):125–130. doi: 10.1128/jb.140.1.125-130.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng Y. S., Zipser D. Purification and characterization of protease III from Escherichia coli. J Biol Chem. 1979 Jun 10;254(11):4698–4706. [PubMed] [Google Scholar]
  9. Clark A. J. Recombination deficient mutants of E. coli and other bacteria. Annu Rev Genet. 1973;7:67–86. doi: 10.1146/annurev.ge.07.120173.000435. [DOI] [PubMed] [Google Scholar]
  10. Davis R., Vapnek D. In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency. J Bacteriol. 1976 Mar;125(3):1148–1155. doi: 10.1128/jb.125.3.1148-1155.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eckhardt T. Use of argA-lac fusions to generate lambda argA-lac bacteriophages and to determine the direction of argA transcription in Escherichia coli. J Bacteriol. 1977 Oct;132(1):60–66. doi: 10.1128/jb.132.1.60-66.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eichler D. C., Lehman I. R. On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli. J Biol Chem. 1977 Jan 25;252(2):499–503. [PubMed] [Google Scholar]
  13. Emmerson P. T., Howard-Flanders P. Cotransduction with thy of a gene required for genetic recombination in Escherichia coli. J Bacteriol. 1967 May;93(5):1729–1731. doi: 10.1128/jb.93.5.1729-1731.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldmark P. J., Linn S. Purification and properties of the recBC DNase of Escherichia coli K-12. J Biol Chem. 1972 Mar 25;247(6):1849–1860. [PubMed] [Google Scholar]
  15. Guyer M. S. The gamma delta sequence of F is an insertion sequence. J Mol Biol. 1978 Dec 15;126(3):347–365. doi: 10.1016/0022-2836(78)90045-1. [DOI] [PubMed] [Google Scholar]
  16. Hickson I. D., Atkinson K. E., Emmerson P. T. Molecular cloning and amplification of the gene for thymidylate synthetase of E. coli. Gene. 1982 Jun;18(3):257–260. doi: 10.1016/0378-1119(82)90163-9. [DOI] [PubMed] [Google Scholar]
  17. Hickson I. D., Emmerson P. T. Identification of the Escherichia coli recB and recC gene products. Nature. 1981 Dec 10;294(5841):578–580. doi: 10.1038/294578a0. [DOI] [PubMed] [Google Scholar]
  18. Howard-Flanders P., Theriot L. Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics. 1966 Jun;53(6):1137–1150. doi: 10.1093/genetics/53.6.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kahn M., Kolter R., Thomas C., Figurski D., Meyer R., Remaut E., Helinski D. R. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Methods Enzymol. 1979;68:268–280. doi: 10.1016/0076-6879(79)68019-9. [DOI] [PubMed] [Google Scholar]
  21. Karn J., Brenner S., Barnett L., Cesareni G. Novel bacteriophage lambda cloning vector. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5172–5176. doi: 10.1073/pnas.77.9.5172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kushner S. R. Differential thermolability of exonuclease and endonuclease activities of the recBC nuclease isolated from thermosensitive recB and recC mutants. J Bacteriol. 1974 Dec;120(3):1219–1222. doi: 10.1128/jb.120.3.1219-1222.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kushner S. R. In vivo studies of temperature-sensitive recB and recC mutants. J Bacteriol. 1974 Dec;120(3):1213–1218. doi: 10.1128/jb.120.3.1213-1218.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lieberman R. P., Oishi M. The recBC deoxyribonuclease of Escherichia coli: isolation and characterization of the subunit proteins and reconstitution of the enzyme. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4816–4820. doi: 10.1073/pnas.71.12.4816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lloyd R. G., Low B., Godson G. N., Birge E. A. Isolation and characterization of an Escherichia coli K-12 mutant with a temperature-sensitive recA- phenotype. J Bacteriol. 1974 Oct;120(1):407–415. doi: 10.1128/jb.120.1.407-415.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  28. Maples V. F., Kushner S. R. DNA repair in Escherichia coli: identification of the uvrD gene product. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5616–5620. doi: 10.1073/pnas.79.18.5616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marvil D. K., Leisinger T. N-acetylglutamate synthase of Escherichia coli: purification, characterization, and molecular properties. J Biol Chem. 1977 May 25;252(10):3295–3303. [PubMed] [Google Scholar]
  30. Muskavitch K. M., Linn S. A unified mechanism for the nuclease and unwinding activities of the recBC enzyme of Escherichia coli. J Biol Chem. 1982 Mar 10;257(5):2641–2648. [PubMed] [Google Scholar]
  31. Nagao R. T., Shah D. M., Eckenrode V. K., Meagher R. B. Multigene family of actin-related sequences isolated from a soybean genomic library. DNA. 1981;1(1):1–9. doi: 10.1089/dna.1.1981.1.1. [DOI] [PubMed] [Google Scholar]
  32. Nossal N. G., Heppel L. A. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem. 1966 Jul 10;241(13):3055–3062. [PubMed] [Google Scholar]
  33. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  34. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sasaki M., Fujiyoshi T., Shimada K., Takagi Y. Fine structure of the recB and recC gene region of Escherichia coli. Biochem Biophys Res Commun. 1982 Nov 30;109(2):414–422. doi: 10.1016/0006-291x(82)91737-5. [DOI] [PubMed] [Google Scholar]
  36. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  37. Swamy K. H., Goldberg A. L. Subcellular distribution of various proteases in Escherichia coli. J Bacteriol. 1982 Mar;149(3):1027–1033. doi: 10.1128/jb.149.3.1027-1033.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tomizawa J., Ogawa H. Structural genes of ATP-dependent deoxyribonuclease of Escherichia coli. Nat New Biol. 1972 Sep 6;239(88):14–16. doi: 10.1038/newbio239014a0. [DOI] [PubMed] [Google Scholar]
  39. Wickner W., Schekman R., Geider K., Kornberg A. A new form of DNA polymerase 3 and a copolymerase replicate a long, single-stranded primer-template. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1764–1767. doi: 10.1073/pnas.70.6.1764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Willetts N. S., Clark A. J. Characteristics of some multiply recombination-deficient strains of Escherichia coli. J Bacteriol. 1969 Oct;100(1):231–239. doi: 10.1128/jb.100.1.231-239.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Willetts N. S., Mount D. W. Genetic analysis of recombination-deficient mutants of Escherichia coli K-12 carrying rec mutations cotransducible with thyA. J Bacteriol. 1969 Nov;100(2):923–934. doi: 10.1128/jb.100.2.923-934.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wu T. T. A model for three-point analysis of random general transduction. Genetics. 1966 Aug;54(2):405–410. doi: 10.1093/genetics/54.2.405. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES