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. 1991 Jun;173(12):3644–3655. doi: 10.1128/jb.173.12.3644-3655.1991

Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis.

J Kooistra 1, G Venema 1
PMCID: PMC207991  PMID: 1646786

Abstract

The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned. The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment. Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-dependent nuclease activity. Three open reading frames were identified on the 8.8-kb SalI-SmaI fragment, which could encode three proteins with molecular masses of 135 (AddB protein), 141 (AddA protein), and 28 kDa. Only the AddB and AddA proteins are required for ATP-dependent exonuclease activity. Both the AddB and AddA proteins contained a conserved amino acid sequence for ATP binding. In the AddA protein, a number of small regions were present showing a high degree of sequence similarity with regions in the E. coli RecB protein. The AddA protein contained six conserved motifs which were also present in the E. coli helicase II (UvrD protein) and the Rep helicase, suggesting that these motifs are involved in the DNA unwinding activity of the enzyme. When linked to the T7 promoter, a high level of expression was obtained in E. coli.

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Selected References

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  1. Ahmed A. A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli. Gene. 1985;39(2-3):305–310. doi: 10.1016/0378-1119(85)90328-2. [DOI] [PubMed] [Google Scholar]
  2. Amundsen S. K., Neiman A. M., Thibodeaux S. M., Smith G. R. Genetic dissection of the biochemical activities of RecBCD enzyme. Genetics. 1990 Sep;126(1):25–40. doi: 10.1093/genetics/126.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amundsen S. K., Taylor A. F., Chaudhury A. M., Smith G. R. recD: the gene for an essential third subunit of exonuclease V. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5558–5562. doi: 10.1073/pnas.83.15.5558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biswal N., Kleinschmidt A. K., Spatz H. C., Trautner T. A. Physical properties of the DNA of bacteriophage SP50. Mol Gen Genet. 1967;100(1):39–55. doi: 10.1007/BF00425774. [DOI] [PubMed] [Google Scholar]
  5. Brendel V., Trifonov E. N. A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res. 1984 May 25;12(10):4411–4427. doi: 10.1093/nar/12.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bron S., Venema G. Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat Res. 1972 May;15(1):1–10. doi: 10.1016/0027-5107(72)90086-3. [DOI] [PubMed] [Google Scholar]
  7. Chao K., Lohman T. M. DNA and nucleotide-induced conformational changes in the Escherichia coli Rep and helicase II (UvrD) proteins. J Biol Chem. 1990 Jan 15;265(2):1067–1076. [PubMed] [Google Scholar]
  8. Chaudhury A. M., Smith G. R. Escherichia coli recBC deletion mutants. J Bacteriol. 1984 Nov;160(2):788–791. doi: 10.1128/jb.160.2.788-791.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng K. C., Smith G. R. Cutting of chi-like sequences by the RecBCD enzyme of Escherichia coli. J Mol Biol. 1987 Apr 20;194(4):747–750. doi: 10.1016/0022-2836(87)90252-x. [DOI] [PubMed] [Google Scholar]
  10. Chestukhin A. V., Shemyakin M. F., Kalinina N. A., Prozorov A. A. Some properties of ATP dependent deoxyribonucleases from normal and rec-mutant strains of Bacillus subtilis. FEBS Lett. 1972 Jul 15;24(1):121–125. doi: 10.1016/0014-5793(72)80841-x. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Doi R. H., Wang L. F. Multiple procaryotic ribonucleic acid polymerase sigma factors. Microbiol Rev. 1986 Sep;50(3):227–243. doi: 10.1128/mr.50.3.227-243.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Doly J., Anagnostopoulos C. Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity. Eur J Biochem. 1976 Dec;71(1):309–316. doi: 10.1111/j.1432-1033.1976.tb11117.x. [DOI] [PubMed] [Google Scholar]
  14. Doly J., Sasarman E., Anagnostopoulos C. ATP-dependent deoxyribonuclease in Bacillus subtilis and a mutant deficient in this activity. Mutat Res. 1974 Jan;22(1):15–23. doi: 10.1016/0027-5107(74)90003-7. [DOI] [PubMed] [Google Scholar]
  15. Finch P. W., Emmerson P. T. The nucleotide sequence of the uvrD gene of E. coli. Nucleic Acids Res. 1984 Jul 25;12(14):5789–5799. doi: 10.1093/nar/12.14.5789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Finch P. W., Storey A., Brown K., Hickson I. D., Emmerson P. T. Complete nucleotide sequence of recD, the structural gene for the alpha subunit of Exonuclease V of Escherichia coli. Nucleic Acids Res. 1986 Nov 11;14(21):8583–8594. doi: 10.1093/nar/14.21.8583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Finch P. W., Storey A., Chapman K. E., Brown K., Hickson I. D., Emmerson P. T. Complete nucleotide sequence of the Escherichia coli recB gene. Nucleic Acids Res. 1986 Nov 11;14(21):8573–8582. doi: 10.1093/nar/14.21.8573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Finch P. W., Wilson R. E., Brown K., Hickson I. D., Emmerson P. T. Complete nucleotide sequence of the Escherichia coli ptr gene encoding protease III. Nucleic Acids Res. 1986 Oct 10;14(19):7695–7703. doi: 10.1093/nar/14.19.7695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Finch P. W., Wilson R. E., Brown K., Hickson I. D., Tomkinson A. E., Emmerson P. T. Complete nucleotide sequence of the Escherichia coli recC gene and of the thyA-recC intergenic region. Nucleic Acids Res. 1986 Jun 11;14(11):4437–4451. doi: 10.1093/nar/14.11.4437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  21. Gilchrist C. A., Denhardt D. T. Escherichia coli rep gene: sequence of the gene, the encoded helicase, and its homology with uvrD. Nucleic Acids Res. 1987 Jan 26;15(2):465–475. doi: 10.1093/nar/15.2.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Hodgman T. C. A new superfamily of replicative proteins. Nature. 1988 May 5;333(6168):22–23. doi: 10.1038/333022b0. [DOI] [PubMed] [Google Scholar]
  25. Iordănescu S. Relationships between cotransducible plasmids in Staphylococcus aureus. J Bacteriol. 1977 Jan;129(1):71–75. doi: 10.1128/jb.129.1.71-75.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Kooistra J., Vosman B., Venema G. Cloning and characterization of a Bacillus subtilis transcription unit involved in ATP-dependent DNase synthesis. J Bacteriol. 1988 Oct;170(10):4791–4797. doi: 10.1128/jb.170.10.4791-4797.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  30. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  31. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  32. Michel B., Niaudet B., Ehrlich S. D. Intermolecular recombination during transformation of Bacillus subtilis competent cells by monomeric and dimeric plasmids. Plasmid. 1983 Jul;10(1):1–10. doi: 10.1016/0147-619x(83)90052-5. [DOI] [PubMed] [Google Scholar]
  33. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  34. Natt E., Scherer G. EMBL 12, a new lambda replacement vector with sites for SalI, XbaI, BamHI, SstI and EcoRI. Nucleic Acids Res. 1986 Sep 11;14(17):7128–7128. [PMC free article] [PubMed] [Google Scholar]
  35. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Palas K. M., Kushner S. R. Biochemical and physical characterization of exonuclease V from Escherichia coli. Comparison of the catalytic activities of the RecBC and RecBCD enzymes. J Biol Chem. 1990 Feb 25;265(6):3447–3454. [PubMed] [Google Scholar]
  38. Peng Z. G., Wu R. A simple and rapid nucleotide sequencing strategy and its application in analyzing a rice histone 3 gene. Gene. 1986;45(3):247–252. doi: 10.1016/0378-1119(86)90022-3. [DOI] [PubMed] [Google Scholar]
  39. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  40. Ponticelli A. S., Schultz D. W., Taylor A. F., Smith G. R. Chi-dependent DNA strand cleavage by RecBC enzyme. Cell. 1985 May;41(1):145–151. doi: 10.1016/0092-8674(85)90069-8. [DOI] [PubMed] [Google Scholar]
  41. Queen C., Korn L. J. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):581–599. doi: 10.1093/nar/12.1part2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Roman L. J., Kowalczykowski S. C. Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. Biochemistry. 1989 Apr 4;28(7):2863–2873. doi: 10.1021/bi00433a018. [DOI] [PubMed] [Google Scholar]
  44. Roman L. J., Kowalczykowski S. C. Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins. J Biol Chem. 1989 Nov 5;264(31):18340–18348. [PubMed] [Google Scholar]
  45. Rosamond J., Telander K. M., Linn S. Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca2+. J Biol Chem. 1979 Sep 10;254(17):8646–8652. [PubMed] [Google Scholar]
  46. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Shemyakin M. F., Grepachevsky A. A., Chestukhin A. V. Properties of Bacillus subtilis ATP-dependent deoxyribonuclease. Eur J Biochem. 1979 Aug 1;98(2):417–423. doi: 10.1111/j.1432-1033.1979.tb13201.x. [DOI] [PubMed] [Google Scholar]
  49. Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. doi: 10.1073/pnas.44.10.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  51. Taylor A. F., Schultz D. W., Ponticelli A. S., Smith G. R. RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting. Cell. 1985 May;41(1):153–163. doi: 10.1016/0092-8674(85)90070-4. [DOI] [PubMed] [Google Scholar]
  52. Taylor A., Smith G. R. Unwinding and rewinding of DNA by the RecBC enzyme. Cell. 1980 Nov;22(2 Pt 2):447–457. doi: 10.1016/0092-8674(80)90355-4. [DOI] [PubMed] [Google Scholar]
  53. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  54. Vosman B., Kooistra J., Olijve J., Venema G. Cloning in Escherichia coli of the gene specifying the DNA-entry nuclease of Bacillus subtilis. Gene. 1987;52(2-3):175–183. doi: 10.1016/0378-1119(87)90044-8. [DOI] [PubMed] [Google Scholar]
  55. Wang T. C., Smith K. C. The roles of RecBCD, Ssb and RecA proteins in the formation of heteroduplexes from linear-duplex DNA in vitro. Mol Gen Genet. 1989 Apr;216(2-3):315–320. doi: 10.1007/BF00334370. [DOI] [PubMed] [Google Scholar]
  56. Wu J. J., Howard M. G., Piggot P. J. Regulation of transcription of the Bacillus subtilis spoIIA locus. J Bacteriol. 1989 Feb;171(2):692–698. doi: 10.1128/jb.171.2.692-698.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yamamoto Y., Ogawa T., Shinagawa H., Nakayama T., Matsuo H., Ogawa H. Determination of the initiation sites of transcription and translation of the uvrD gene of Escherichia coli. J Biochem. 1986 Jun;99(6):1579–1590. doi: 10.1093/oxfordjournals.jbchem.a135631. [DOI] [PubMed] [Google Scholar]
  58. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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