Skip to main content
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 Mar;71(3):947–951. doi: 10.1073/pnas.71.3.947

Photochemical Attachment of lac Repressor to Bromodeoxyuridine-Substituted lac Operator by Ultraviolet Radiation

Syr-Yaung Lin 1, Arthur D Riggs 1
PMCID: PMC388134  PMID: 4522804

Abstract

The transducing phage λh80dlac carries the lac operator, whereas wild-type λh80 does not. We find that in high salt (0.18 M KCl), ultraviolet radiation causes the formation of a very stable complex between repressor and 5-bromodeoxyuridine (BrdU)-substituted λh80dlac but not to BrdU-λh80 DNA. Studies with inducers of the lac operon confirm the specificity of attachment. In low slat (0.01 M KCl), ultraviolet radiation will also attach repressor nonspecifically to BrdU-λh80 DNA. The stability of the complex suggests that covalent bonds are formed. We also report that another regulatory protein, the catabolite gene activator protein, can be attached similarly to DNA.

Keywords: catabolite gene activator protein, protein-DNA interaction, nitrocellulose-filter assay

Full text

PDF
947

Selected References

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

  1. Bourgeois S., Riggs A. D. The lac repressor-operator interaction. IV. Assay and purification of operator DNA. Biochem Biophys Res Commun. 1970 Jan 23;38(2):348–354. doi: 10.1016/0006-291x(70)90719-9. [DOI] [PubMed] [Google Scholar]
  2. Gilbert W., Müller-Hill B. Isolation of the lac repressor. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1891–1898. doi: 10.1073/pnas.56.6.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gilbert W., Müller-Hill B. The lac operator is DNA. Proc Natl Acad Sci U S A. 1967 Dec;58(6):2415–2421. doi: 10.1073/pnas.58.6.2415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hackett P., Jr, Hanawalt P. Selectivity for thymine over 5-bromouracil by a thymine-requiring bacterium. Biochim Biophys Acta. 1966 Aug 17;123(2):356–363. doi: 10.1016/0005-2787(66)90288-7. [DOI] [PubMed] [Google Scholar]
  5. Jobe A., Bourgeois S. Lac repressor-operator interaction. 8. Lactose is an anti-inducer of the lac operon. J Mol Biol. 1973 Apr 5;75(2):303–313. doi: 10.1016/0022-2836(73)90023-5. [DOI] [PubMed] [Google Scholar]
  6. Jobe A., Riggs A. D., Bourgeois S. Lac repressor-operator interaction. V. Characterization of super- and pseudo-wild-type repressors. J Mol Biol. 1972 Feb 28;64(1):181–199. doi: 10.1016/0022-2836(72)90328-2. [DOI] [PubMed] [Google Scholar]
  7. Laiken S. L., Gross C. A., Von Hippel P. H. Equilibrium and kinetic studies of Escherichia coli lac repressor-inducer interactions. J Mol Biol. 1972 Apr 28;66(1):143–155. doi: 10.1016/s0022-2836(72)80012-3. [DOI] [PubMed] [Google Scholar]
  8. Lin S. Y., Riggs A. D. Lac operator analogues: bromodeoxyuridine substitution in the lac operator affects the rate of dissociation of the lac repressor. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2574–2576. doi: 10.1073/pnas.69.9.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lin S. Y., Riggs A. D. Lac repressor binding to non-operator DNA: detailed studies and a comparison of eequilibrium and rate competition methods. J Mol Biol. 1972 Dec 30;72(3):671–690. doi: 10.1016/0022-2836(72)90184-2. [DOI] [PubMed] [Google Scholar]
  10. Lin S. Y., Riggs A. D. Lac repressor binding to operator analogues: comparison of poly(d(A-T)), poly(d(A-BrU)), and poly(d(A-U)). Biochem Biophys Res Commun. 1971 Dec 17;45(6):1542–1547. doi: 10.1016/0006-291x(71)90195-1. [DOI] [PubMed] [Google Scholar]
  11. Markovitz A. Ultraviolet light-induced stable complexes of DNA and DNA polymerase. Biochim Biophys Acta. 1972 Nov 9;281(4):522–534. doi: 10.1016/0005-2787(72)90153-0. [DOI] [PubMed] [Google Scholar]
  12. Matthews K. S., Matthews H. R., Thielmann H. W., Jardetzky G. Ultraviolet difference spectra of the lactose repressor protein. Biochim Biophys Acta. 1973 Jan 25;295(1):159–165. doi: 10.1016/0005-2795(73)90083-4. [DOI] [PubMed] [Google Scholar]
  13. Nissley P., Anderson W. B., Gallo M., Pastan I., Perlman R. L. The binding of cyclic adenosine monophosphate receptor to deoxyribonucleic acid. J Biol Chem. 1972 Jul 10;247(13):4264–4269. [PubMed] [Google Scholar]
  14. Oshima Y., Matsuura M., Horiuchi T. Conformational change of the lac repressor induced with the inducer. Biochem Biophys Res Commun. 1972 Jun 28;47(6):1444–1450. doi: 10.1016/0006-291x(72)90234-3. [DOI] [PubMed] [Google Scholar]
  15. Riggs A. D., Bourgeois S., Cohn M. The lac repressor-operator interaction. 3. Kinetic studies. J Mol Biol. 1970 Nov 14;53(3):401–417. doi: 10.1016/0022-2836(70)90074-4. [DOI] [PubMed] [Google Scholar]
  16. Riggs A. D., Bourgeois S., Newby R. F., Cohn M. DNA binding of the lac repressor. J Mol Biol. 1968 Jul 14;34(2):365–368. doi: 10.1016/0022-2836(68)90261-1. [DOI] [PubMed] [Google Scholar]
  17. Riggs A. D., Newby R. F., Bourgeois S. lac repressor--operator interaction. II. Effect of galactosides and other ligands. J Mol Biol. 1970 Jul 28;51(2):303–314. doi: 10.1016/0022-2836(70)90144-0. [DOI] [PubMed] [Google Scholar]
  18. Riggs A. D., Reiness G., Zubay G. Purification and DNA-binding properties of the catabolite gene activator protein. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1222–1225. doi: 10.1073/pnas.68.6.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
  20. SMITH K. C. Dose dependent decrease in extractability of DNA from bacteria following irradiation with ultraviolet light or with visible light plus dye. Biochem Biophys Res Commun. 1962 Jul 3;8:157–163. doi: 10.1016/0006-291x(62)90255-3. [DOI] [PubMed] [Google Scholar]
  21. Setlow R. B., Setlow J. K. Effects of radiation on polynucleotides. Annu Rev Biophys Bioeng. 1972;1:293–346. doi: 10.1146/annurev.bb.01.060172.001453. [DOI] [PubMed] [Google Scholar]
  22. Smets L. A., Cornelis J. J. Repairable and irrepairable damage in 5-bromouracil-substituted DNA exposed to ultra-violet radiation. Int J Radiat Biol Relat Stud Phys Chem Med. 1971;19(5):445–457. doi: 10.1080/09553007114550581. [DOI] [PubMed] [Google Scholar]
  23. Smith K. C., Aplin R. T. A mixed photoproduct of uracil and cysteine (5-S-cysteine-6-hydrouracil). A possible model for the in vivo cross-linking of deoxyribonucleic acid and protein by ultraviolet light. Biochemistry. 1966 Jun;5(6):2125–2130. doi: 10.1021/bi00870a046. [DOI] [PubMed] [Google Scholar]
  24. Smith K. C., Meun D. H. Kinetics of the photochemical addition of [35S] cysteine to polynucleotides and nucleic acids. Biochemistry. 1968 Mar;7(3):1033–1037. doi: 10.1021/bi00843a023. [DOI] [PubMed] [Google Scholar]
  25. Smith K. C. Photochemical addition of amino acids to 14C-uracil. Biochem Biophys Res Commun. 1969 Feb 7;34(3):354–357. doi: 10.1016/0006-291x(69)90840-7. [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