Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1974 Sep;71(9):3593–3597. doi: 10.1073/pnas.71.9.3593

Isolation of Exonuclease VIII: The Enzyme Associated with the sbcA Indirect Suppressor

Sidney R Kushner *,†, Haruko Nagaishi ‡, A J Clark ‡
PMCID: PMC433821  PMID: 4610579

Abstract

recB and/or recC deficiency in Escherichia coli K-12 is indirectly suppressed by the presence of sbcA- mutations. sbcA- strains contain an increased level of an ATP-independent nuclease. Genetic and enzymatic tests indicate that this activity is not exonuclease III, exonuclease V (recB-recC nuclease), DNA polymerase I, or lambda exonuclease. This new enzyme (exonuclease VIII) has been purified 750-fold and shows a striking preference for double-stranded DNA over heat-denatured DNA. It does not act endonucleolytically on closed circular, single-stranded DNA as exonuclease V does. It also lacks a 3′-phosphatase function. Analysis on sodium dodecyl sulfate-polyacrylamide gels indicates that exonuclease VIII is not present in unsuppressed (sbcA+) strains. It is thought that sbcA determines some type of control function; the structural gene for exonuclease VIII is denoted by recE.

Keywords: genetic recombination, Escherichia coli, recB, exonuclease V

Full text

PDF
3593

Selected References

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

  1. Barbour S. D., Nagaishi H., Templin A., Clark A. J. Biochemical and genetic studies of recombination proficiency in Escherichia coli. II. Rec+ revertants caused by indirect suppression of rec- mutations. Proc Natl Acad Sci U S A. 1970 Sep;67(1):128–135. doi: 10.1073/pnas.67.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carter D. M., Radding C. M. The role of exonuclease and beta protein of phage lambda in genetic recombination. II. Substrate specificity and the mode of action of lambda exonuclease. J Biol Chem. 1971 Apr 25;246(8):2502–2512. [PubMed] [Google Scholar]
  3. 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]
  4. Clark A. J. The beginning of a genetic analysis of recombination proficiency. J Cell Physiol. 1967 Oct;70(2 Suppl):165–180. doi: 10.1002/jcp.1040700412. [DOI] [PubMed] [Google Scholar]
  5. Cozzarelli N. R., Kelly R. B., Kornberg A. Enzymic synthesis of DNA. 33. Hydrolysis of a 5'-triphosphate-terminated polynucleotide in the active center of DNA polymerase. J Mol Biol. 1969 Nov 14;45(3):513–531. doi: 10.1016/0022-2836(69)90309-x. [DOI] [PubMed] [Google Scholar]
  6. Forsheit A. B., Ray D. S. Conformations of the single-stranded DNA of bacteriophage M13. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1534–1541. doi: 10.1073/pnas.67.3.1534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Hayes D. H., Hayes F., Guérin M. F. Association of rapidly labelled bacterial RNA with ribosomal RNA in solutions of high ionic strength. II. J Mol Biol. 1966 Jul;18(3):499–515. doi: 10.1016/s0022-2836(66)80039-6. [DOI] [PubMed] [Google Scholar]
  9. Horii Z., Clark A. J. Genetic analysis of the recF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J Mol Biol. 1973 Oct 25;80(2):327–344. doi: 10.1016/0022-2836(73)90176-9. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Kushner S. R., Nagaishi H., Templin A., Clark A. J. Genetic recombination in Escherichia coli: the role of exonuclease I. Proc Natl Acad Sci U S A. 1971 Apr;68(4):824–827. doi: 10.1073/pnas.68.4.824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Little J. W., Lehman I. R., Kaiser A. D. An exonuclease induced by bacteriophage lambda. I. Preparation of the crystalline enzyme. J Biol Chem. 1967 Feb 25;242(4):672–678. [PubMed] [Google Scholar]
  14. Low B. Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K12. Proc Natl Acad Sci U S A. 1968 May;60(1):160–167. doi: 10.1073/pnas.60.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Milcarek C., Weiss B. Mutants of Escherichia coli with altered deoxyribonucleases. I. Isolation and characterization of mutants for exonuclease 3. J Mol Biol. 1972 Jul 21;68(2):303–318. doi: 10.1016/0022-2836(72)90215-x. [DOI] [PubMed] [Google Scholar]
  16. RICHARDSON C. C., KORNBERG A. A DEOXYRIBONUCLEIC ACID PHOSPHATASE-EXONUCLEASE FROM ESCHERICHIA COLI. I. PURIFICATION OF THE ENZYME AND CHARACTERIZATION OF THE PHOSPHATASE ACTIVITY. J Biol Chem. 1964 Jan;239:242–250. [PubMed] [Google Scholar]
  17. RICHARDSON C. C., SCHILDKRAUT C. L., APOSHIAN H. V., KORNBERG A. ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. XIV. FURTHER PURIFICATION AND PROPERTIES OF DEOXYRIBONUCLEIC ACID POLYMERASE OF ESCHERICHIA COLI. J Biol Chem. 1964 Jan;239:222–232. [PubMed] [Google Scholar]
  18. Templin A., Kushner S. R., Clark A. J. Genetic analysis of mutations indirectly suppressing recB and recC mutations. Genetics. 1972 Oct;72(2):105–115. [PMC free article] [PubMed] [Google Scholar]
  19. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  20. Willetts N. S., Clark A. J., Low B. Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J Bacteriol. 1969 Jan;97(1):244–249. doi: 10.1128/jb.97.1.244-249.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES