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. 1989 May;171(5):2278–2286. doi: 10.1128/jb.171.5.2278-2286.1989

The exoA gene of Streptococcus pneumoniae and its product, a DNA exonuclease with apurinic endonuclease activity.

A Puyet 1, B Greenberg 1, S A Lacks 1
PMCID: PMC209899  PMID: 2468646

Abstract

The gene encoding the major DNA exonuclease of Streptococcus pneumoniae, exoA, was cloned in a streptococcal host vector system. Its location was determined by subcloning and by insertion mutations. Transfer of a DNA segment containing the gene to an Escherichia coli expression vector showed that exoA was the structural gene for the enzyme and that it was adjacent to its promoter. DNA sequence determination indicated that the gene encoded a protein, ExoA, of molecular weight 31,263. Under hyperexpression conditions, the ExoA protein constituted 10% of total cellular protein. In addition to previously demonstrated 3' to 5' exonuclease and 3'-phosphatase activities, ExoA was shown to make single-strand breaks at apurinic sites in DNA. Its enzymatic activities are thus similar to those of exonuclease III of E. coli and other gram-negative bacteria. The nucleotide sequence of exoA revealed it to be homologous to xth of E. coli, with 26% identity of amino acid residues in the predicted proteins. So far, no null chromosomal mutants of exoA have been obtained, and the biological function of ExoA remains unknown.

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Selected References

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  1. BERNS K. I., THOMAS C. A., Jr ISOLATION OF HIGH MOLECULAR WEIGHT DNA FROM HEMOPHILUS INFLUENZAE. J Mol Biol. 1965 Mar;11:476–490. doi: 10.1016/s0022-2836(65)80004-3. [DOI] [PubMed] [Google Scholar]
  2. Balganesh T. S., Lacks S. A. Plasmid vector for cloning in Streptococcus pneumoniae and strategies for enrichment for recombinant plasmids. Gene. 1984 Jul-Aug;29(1-2):221–230. doi: 10.1016/0378-1119(84)90182-3. [DOI] [PubMed] [Google Scholar]
  3. Ballester S., Lopez P., Alonso J. C., Espinosa M., Lacks S. A. Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids. Gene. 1986;41(2-3):153–163. doi: 10.1016/0378-1119(86)90094-6. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chen J. D., Morrison D. A. Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene. 1987;55(2-3):179–187. doi: 10.1016/0378-1119(87)90278-2. [DOI] [PubMed] [Google Scholar]
  6. Clements J. E., Rogers S. G., Weiss B. A DNase for apurinic/apyrimidinic sites associated with exonuclease III of Hemophilus influenzae. J Biol Chem. 1978 May 10;253(9):2990–2999. [PubMed] [Google Scholar]
  7. Coughlin S. A., Green D. M. Two-dimensional zymogram analysis of nucleases in Bacillus subtilis. Anal Biochem. 1983 Sep;133(2):322–329. doi: 10.1016/0003-2697(83)90091-x. [DOI] [PubMed] [Google Scholar]
  8. Cunningham R. P., Saporito S. M., Spitzer S. G., Weiss B. Endonuclease IV (nfo) mutant of Escherichia coli. J Bacteriol. 1986 Dec;168(3):1120–1127. doi: 10.1128/jb.168.3.1120-1127.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Currier T. C., Nester E. W. Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal Biochem. 1976 Dec;76(2):431–441. doi: 10.1016/0003-2697(76)90338-9. [DOI] [PubMed] [Google Scholar]
  10. Gunther J. K., Goodgal S. H. An exonuclease specific for double stranded deoxyribonucleic acid. J Biol Chem. 1970 Oct 25;245(20):5341–5349. [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. LAWLEY P. D., BROOKES P. FURTHER STUDIES ON THE ALKYLATION OF NUCLEIC ACIDS AND THEIR CONSTITUENT NUCLEOTIDES. Biochem J. 1963 Oct;89:127–138. doi: 10.1042/bj0890127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Lacks S. A., Lopez P., Greenberg B., Espinosa M. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J Mol Biol. 1986 Dec 20;192(4):753–765. doi: 10.1016/0022-2836(86)90026-4. [DOI] [PubMed] [Google Scholar]
  15. Lacks S. A., Springhorn S. S. Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase. J Bacteriol. 1984 Mar;157(3):934–936. doi: 10.1128/jb.157.3.934-936.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lacks S. A., Springhorn S. S., Rosenthal A. L. Effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis. Anal Biochem. 1979 Dec;100(2):357–363. doi: 10.1016/0003-2697(79)90241-0. [DOI] [PubMed] [Google Scholar]
  17. Lacks S., Greenberg B. A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA. J Biol Chem. 1975 Jun 10;250(11):4060–4066. [PubMed] [Google Scholar]
  18. Lacks S., Greenberg B. Deoxyribonucleases of Pneumococcus. J Biol Chem. 1967 Jul 10;242(13):3108–3120. [PubMed] [Google Scholar]
  19. Lacks S., Greenberg B., Neuberger M. Identification of a deoxyribonuclease implicated in genetic transformation of Diplococcus pneumoniae. J Bacteriol. 1975 Jul;123(1):222–232. doi: 10.1128/jb.123.1.222-232.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lacks S., Greenberg B., Neuberger M. Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2305–2309. doi: 10.1073/pnas.71.6.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lacks S., Greenberg B. Single-strand breakage on binding of DNA to cells in the genetic transformation of Diplococcus pneumoniae. J Mol Biol. 1976 Feb 25;101(2):255–275. doi: 10.1016/0022-2836(76)90376-4. [DOI] [PubMed] [Google Scholar]
  22. Lacks S. Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and other activities possibly pertinent to genetic transformation. J Bacteriol. 1970 Feb;101(2):373–383. doi: 10.1128/jb.101.2.373-383.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lacks S., Neuberger M. Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae. J Bacteriol. 1975 Dec;124(3):1321–1329. doi: 10.1128/jb.124.3.1321-1329.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lindahl T. An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3649–3653. doi: 10.1073/pnas.71.9.3649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lindler L. E., Macrina F. L. Molecular cloning and characterization of a Streptococcus sanguis DNase necessary for repair of DNA damage induced by UV light and methyl methanesulfonate. J Bacteriol. 1987 Jul;169(7):3199–3208. doi: 10.1128/jb.169.7.3199-3208.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lopez P., Espinosa M., Lacks S. A. Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis. Mol Gen Genet. 1984;195(3):403–410. doi: 10.1007/BF00341440. [DOI] [PubMed] [Google Scholar]
  27. Lopez P., Espinosa M., Stassi D. L., Lacks S. A. Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology. J Bacteriol. 1982 May;150(2):692–701. doi: 10.1128/jb.150.2.692-701.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lopez P., Martinez S., Diaz A., Espinosa M., Lacks S. A. Characterization of the polA gene of Streptococcus pneumoniae and comparison of the DNA polymerase I it encodes to homologous enzymes from Escherichia coli and phage T7. J Biol Chem. 1989 Mar 5;264(7):4255–4263. [PubMed] [Google Scholar]
  29. Mannarelli B. M., Balganesh T. S., Greenberg B., Springhorn S. S., Lacks S. A. Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4468–4472. doi: 10.1073/pnas.82.13.4468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Martinez S., Lopez P., Espinosa M., Lacks S. A. Cloning of a gene encoding a DNA polymerase-exonuclease of Streptococcus pneumoniae. Gene. 1986;44(1):79–88. doi: 10.1016/0378-1119(86)90045-4. [DOI] [PubMed] [Google Scholar]
  31. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  32. Muckerman C. C., Springhorn S. S., Greenberg B., Lacks S. A. Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae. J Bacteriol. 1982 Oct;152(1):183–190. doi: 10.1128/jb.152.1.183-190.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Priebe S. D., Hadi S. M., Greenberg B., Lacks S. A. Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium. J Bacteriol. 1988 Jan;170(1):190–196. doi: 10.1128/jb.170.1.190-196.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. RICHARDSON C. C., LEHMAN I. R., KORNBERG A. A DEOXYRIBONUCLEIC ACID PHOSPHATASE-EXONUCLEASE FROM ESCHERICHIA COLI. II. CHARACTERIZATION OF THE EXONUCLEASE ACTIVITY. J Biol Chem. 1964 Jan;239:251–258. [PubMed] [Google Scholar]
  35. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  36. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  37. Rosenthal A. L., Lacks S. A. Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal Biochem. 1977 May 15;80(1):76–90. doi: 10.1016/0003-2697(77)90627-3. [DOI] [PubMed] [Google Scholar]
  38. Saporito S. M., Cunningham R. P. Nucleotide sequence of the nfo gene of Escherichia coli K-12. J Bacteriol. 1988 Nov;170(11):5141–5145. doi: 10.1128/jb.170.11.5141-5145.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Saporito S. M., Smith-White B. J., Cunningham R. P. Nucleotide sequence of the xth gene of Escherichia coli K-12. J Bacteriol. 1988 Oct;170(10):4542–4547. doi: 10.1128/jb.170.10.4542-4547.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  41. Stassi D. L., Lopez P., Espinosa M., Lacks S. A. Cloning of chromosomal genes in Streptococcus pneumoniae. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7028–7032. doi: 10.1073/pnas.78.11.7028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Taylor A. F., Weiss B. Role of exonuclease III in the base excision repair of uracil-containing DNA. J Bacteriol. 1982 Jul;151(1):351–357. doi: 10.1128/jb.151.1.351-357.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tiemeier D. C., Tilghman S. M., Leder P. Purification and cloning of a mouse ribosomal gene fragment in coliphage lambda. Gene. 1977;2(3-4):173–191. doi: 10.1016/0378-1119(77)90016-6. [DOI] [PubMed] [Google Scholar]
  45. Weiss B. Endonuclease II of Escherichia coli is exonuclease III. J Biol Chem. 1976 Apr 10;251(7):1896–1901. [PubMed] [Google Scholar]
  46. de la Campa A. G., Kale P., Springhorn S. S., Lacks S. A. Proteins encoded by the DpnII restriction gene cassette. Two methylases and an endonuclease. J Mol Biol. 1987 Aug 5;196(3):457–469. doi: 10.1016/0022-2836(87)90024-6. [DOI] [PubMed] [Google Scholar]

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