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
. 1973 Aug;70(8):2215–2219. doi: 10.1073/pnas.70.8.2215

Purification and Properties of the γ-Protein Specified by Bacteriophage λ: An Inhibitor of the Host RecBC Recombination Enzyme

Yoshiyuki Sakaki 1,2, Alexander E Karu 1,2, Stuart Linn 1,2, Harrison Echols 1,2
PMCID: PMC433704  PMID: 4275917

Abstract

Previous experiments have indicated that the gam gene of bacteriophage λ is responsible for an inhibition of the RecBC DNase—an enzyme that is essential for the major host pathway of genetic recombination. We report here experiments that define the inhibitor as the protein product of the gam gene (“γ-protein”) and that characterize the inhibition reaction with highly purified preparations of γ-protein and RecBC DNase. Genetic characterization was performed with partially purified fractions prepared from cells infected with various λ mutants. An activity that inhibits RecBC DNase was absent in extracts prepared after infection by phage that carry nonsense or deletion mutations in the gam gene; this activity was highly thermolabile in an extract prepared after infection by phage that carry a temperature-sensitive mutation in the gam gene. For biochemical characterization, the γ-protein has been purified more than 800-fold. This highly purified preparation inhibited all of the known catalytic activities associated with the RecBC enzyme, but exhibited no detectable DNase or ATPase activities by itself. These findings are discussed in terms of their implications for regulation of genetic recombination and bacteriophage λ development.

Keywords: bacteriophage λ gam gene, RecBC DNase, exonuclease V

Full text

PDF
2215

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. Clark A. J. Toward a metabolic interpretation of genetic recombination of E. coli and its phages. Annu Rev Microbiol. 1971;25:437–464. doi: 10.1146/annurev.mi.25.100171.002253. [DOI] [PubMed] [Google Scholar]
  3. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  4. Enquist L. W., Skalka A. Replication of bacteriophage lambda DNA dependent on the function of host and viral genes. I. Interaction of red, gam and rec. J Mol Biol. 1973 Apr 5;75(2):185–212. doi: 10.1016/0022-2836(73)90016-8. [DOI] [PubMed] [Google Scholar]
  5. Eskin B., Linn S. The deoxyribonucleic acid modification and restriction enzymes of Escherichia coli B. II. Purification, subunit structure, and catalytic properties of the restriction endonuclease. J Biol Chem. 1972 Oct 10;247(19):6183–6191. [PubMed] [Google Scholar]
  6. 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]
  7. Israel V., Woodworth-Gutai M., Levine M. Inhibitory effect of bacteriophage P22 infection on host cell deoxyribonuclease activity. J Virol. 1972 May;9(5):752–757. doi: 10.1128/jvi.9.5.752-757.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Karu A. E., Linn S. Uncoupling of the recBC ATPase from DNase by DNA crosslinked with psoralen. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2855–2859. doi: 10.1073/pnas.69.10.2855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LEHMAN I. R., NUSSBAUM A. L. THE DEOXYRIBONUCLEASES OF ESCHERICHIA COLI. V. ON THE SPECIFICITY OF EXONUCLEASE I (PHOSPHODIESTERASE). J Biol Chem. 1964 Aug;239:2628–2636. [PubMed] [Google Scholar]
  11. LEHMAN I. R. The deoxyribonucleases of Escherichia coli. I. Purification and properties of a phosphodiesterase. J Biol Chem. 1960 May;235:1479–1487. [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. Lindahl G., Sironi G., Bialy H., Calendar R. Bacteriophage lambda; abortive infection of bacteria lysogenic for phage P2. Proc Natl Acad Sci U S A. 1970 Jul;66(3):587–594. doi: 10.1073/pnas.66.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Pero J. Location of the phage lambda gene responsible for turning off lambda-exonuclease synthesis. Virology. 1970 Jan;40(1):65–71. doi: 10.1016/0042-6822(70)90379-x. [DOI] [PubMed] [Google Scholar]
  16. Pilarksi L. M., Egan J. B. Role of DNA topology in transcription of coliphage lambda in vivo. II. DNA topology protects the template from exonuclease attack. J Mol Biol. 1973 May 15;76(2):257–266. doi: 10.1016/0022-2836(73)90389-6. [DOI] [PubMed] [Google Scholar]
  17. RADDING C. M. NUCLEASE ACTIVITY IN DEFECTIVE LYSOGENS OF PHAGE MU. II. A HYPERACTIVE MUTANT. Proc Natl Acad Sci U S A. 1964 Oct;52:965–973. doi: 10.1073/pnas.52.4.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Richardson C. C. The 5'-terminal nucleotides of T7 bacteriophage deoxyribonucleic acid. J Mol Biol. 1966 Jan;15(1):49–61. doi: 10.1016/s0022-2836(66)80208-5. [DOI] [PubMed] [Google Scholar]
  19. SUSSMAN R., JACOB F. [On a thermosensitive repression system in the Escherichia coli lambda bacteriophage]. C R Hebd Seances Acad Sci. 1962 Feb 19;254:1517–1519. [PubMed] [Google Scholar]
  20. Shulman M. J., Hallick L. M., Echols H., Signer E. R. Properties of recombination-deficient mutants of bacteriophage lambda. J Mol Biol. 1970 Sep 28;52(3):501–520. doi: 10.1016/0022-2836(70)90416-x. [DOI] [PubMed] [Google Scholar]
  21. Sironi G., Bialy H., Lozeron H. A., Calendar R. Bacteriophage P2: interaction with phage lambda and with recombination-deficient bacteria. Virology. 1971 Nov;46(2):387–396. doi: 10.1016/0042-6822(71)90040-7. [DOI] [PubMed] [Google Scholar]
  22. Stahl F. W., McMilin K. D., Stahl M. M., Malone R. E., Nozu Y., Russo V. E. A role for recombination in the production of "free-loader" lambda bacteriophage particles. J Mol Biol. 1972 Jul 14;68(1):57–67. doi: 10.1016/0022-2836(72)90262-8. [DOI] [PubMed] [Google Scholar]
  23. Tanner D., Oishi M. The effect of bacteriophage T4 infection on an ATP-dependent deoxyribonuclease in Escherichia coli. Biochim Biophys Acta. 1971 Feb 11;228(3):767–769. doi: 10.1016/0005-2787(71)90747-7. [DOI] [PubMed] [Google Scholar]
  24. Unger R. C., Clark A. J. Interaction of the recombination pathways of bacteriophage lambda and its host Escherichia coli K12: effects on exonuclease V activity. J Mol Biol. 1972 Oct 14;70(3):539–548. doi: 10.1016/0022-2836(72)90558-x. [DOI] [PubMed] [Google Scholar]
  25. Unger R. C., Echols H., Clark A. J. Interaction of the recombination pathways of bacteriophage lambda and host Escherichia coli: effects on lambda recombination. J Mol Biol. 1972 Oct 14;70(3):531–537. doi: 10.1016/0022-2836(72)90557-8. [DOI] [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