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. 1986 Oct;83(19):7147–7151. doi: 10.1073/pnas.83.19.7147

Nuclease activity of 1,10-phenanthroline-copper: sequence-specific targeting.

C H Chen, D S Sigman
PMCID: PMC386672  PMID: 3020536

Abstract

The nuclease activity of 1,10-phenanthroline-copper ion can be targeted to specific DNA sequences by attachment of the ligand to the 5' end of complementary deoxyoligonucleotides via a phosphoramidate linkage. To synthesize the adduct, the phosphorimidazolide of the deoxyoligonucleotide is prepared using a water-soluble carbodiimide and is then coupled to 5-glycylamido-1,10-phenanthroline. After hybridization to the target DNA, sequence-specific cleavage is observed upon the addition of cupric ion and 3-mercaptopropionic acid. Two methods of assaying the cutting of the operator sequence of the lac operon have been employed using the oligonucleotide 5'-AATTGTTATCCGCTCACAATT-3' representing sequence positions 21-1 of the template strand. In the first, the single-stranded DNA of the phage M13mp8 was the target, and cuts were detected using a primer-extension assay. In the second, the substrate was an EcoRI fragment 3' labeled in the nontemplate strand. After denaturation and reannealing to the oligonucleotide-1,10-phenanthroline adduct, cupric ion and 3-mercaptopropionic acid were added, and the products were analyzed directly on a sequencing gel. With the phenanthroline moiety attached to position 21 of the oligonucleotide carrier, cutting was observed at positions 20-25 using both assays.

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Selected References

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

  1. Cartwright I. L., Hertzberg R. P., Dervan P. B., Elgin S. C. Cleavage of chromatin with methidiumpropyl-EDTA . iron(II). Proc Natl Acad Sci U S A. 1983 Jun;80(11):3213–3217. doi: 10.1073/pnas.80.11.3213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chu B. C., Orgel L. E. Nonenzymatic sequence-specific cleavage of single-stranded DNA. Proc Natl Acad Sci U S A. 1985 Feb;82(4):963–967. doi: 10.1073/pnas.82.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chu B. C., Wahl G. M., Orgel L. E. Derivatization of unprotected polynucleotides. Nucleic Acids Res. 1983 Sep 24;11(18):6513–6529. doi: 10.1093/nar/11.18.6513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. D'Aurora V., Stern A. M., Sigman D. S. Inhibition of E. coli DNA polymerase I by 1,10-phenanthroline. Biochem Biophys Res Commun. 1977 Sep 9;78(1):170–176. doi: 10.1016/0006-291x(77)91236-0. [DOI] [PubMed] [Google Scholar]
  5. Dreyer G. B., Dervan P. B. Sequence-specific cleavage of single-stranded DNA: oligodeoxynucleotide-EDTA X Fe(II). Proc Natl Acad Sci U S A. 1985 Feb;82(4):968–972. doi: 10.1073/pnas.82.4.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gralla J. D. Rapid "footprinting" on supercoiled DNA. Proc Natl Acad Sci U S A. 1985 May;82(10):3078–3081. doi: 10.1073/pnas.82.10.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Henner W. D., Grunberg S. M., Haseltine W. A. Sites and structure of gamma radiation-induced DNA strand breaks. J Biol Chem. 1982 Oct 10;257(19):11750–11754. [PubMed] [Google Scholar]
  8. Henner W. D., Rodriguez L. O., Hecht S. M., Haseltine W. A. gamma Ray induced deoxyribonucleic acid strand breaks. 3' Glycolate termini. J Biol Chem. 1983 Jan 25;258(2):711–713. [PubMed] [Google Scholar]
  9. Hertzberg R. P., Dervan P. B. Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses. Biochemistry. 1984 Aug 14;23(17):3934–3945. doi: 10.1021/bi00312a022. [DOI] [PubMed] [Google Scholar]
  10. Jen-Jacobson L., Kurpiewski M., Lesser D., Grable J., Boyer H. W., Rosenberg J. M., Greene P. J. Coordinate ion pair formation between EcoRI endonuclease and DNA. J Biol Chem. 1983 Dec 10;258(23):14638–14646. [PubMed] [Google Scholar]
  11. Jessee B., Gargiulo G., Razvi F., Worcel A. Analogous cleavage of DNA by micrococcal nuclease and a 1-10-phenanthroline-cuprous complex. Nucleic Acids Res. 1982 Oct 11;10(19):5823–5834. doi: 10.1093/nar/10.19.5823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jessee B., Gargiulo G., Razvi F., Worcel A. Analogous cleavage of DNA by micrococcal nuclease and a 1-10-phenanthroline-cuprous complex. Nucleic Acids Res. 1982 Oct 11;10(19):5823–5834. doi: 10.1093/nar/10.19.5823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kopka M. L., Yoon C., Goodsell D., Pjura P., Dickerson R. E. Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G. J Mol Biol. 1985 Jun 25;183(4):553–563. doi: 10.1016/0022-2836(85)90171-8. [DOI] [PubMed] [Google Scholar]
  14. Kopka M. L., Yoon C., Goodsell D., Pjura P., Dickerson R. E. The molecular origin of DNA-drug specificity in netropsin and distamycin. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1376–1380. doi: 10.1073/pnas.82.5.1376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lawson T. G., Regnier F. E., Weith H. L. Separation of synthetic oligonucleotides on columns of microparticulate silica coated with crosslinked polyethylene imine. Anal Biochem. 1983 Aug;133(1):85–93. doi: 10.1016/0003-2697(83)90225-7. [DOI] [PubMed] [Google Scholar]
  16. Marshall L. E., Graham D. R., Reich K. A., Sigman D. S. Cleavage of deoxyribonucleic acid by the 1,10-phenanthroline-cuprous complex. Hydrogen peroxide requirement and primary and secondary structure specificity. Biochemistry. 1981 Jan 20;20(2):244–250. doi: 10.1021/bi00505a003. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  20. Pope L. E., Sigman D. S. Secondary structure specificity of the nuclease activity of the 1,10-phenanthroline-copper complex. Proc Natl Acad Sci U S A. 1984 Jan;81(1):3–7. doi: 10.1073/pnas.81.1.3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pope L. M., Reich K. A., Graham D. R., Sigman D. S. Products of DNA cleavage by the 1,10-phenanthroline-copper complex. Inhibitors of Escherichia coli DNA polymerase I. J Biol Chem. 1982 Oct 25;257(20):12121–12128. [PubMed] [Google Scholar]
  22. Pribnow D. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci U S A. 1975 Mar;72(3):784–788. doi: 10.1073/pnas.72.3.784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sigman D. S., Graham D. R., D'Aurora V., Stern A. M. Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline . cuprous complex. Inhibition of Escherichia coli DNA polymerase I. J Biol Chem. 1979 Dec 25;254(24):12269–12272. [PubMed] [Google Scholar]
  24. Sigman D. S., Spassky A., Rimsky S., Buc H. Conformational analysis of lac promoters using the nuclease activity of 1,10-phenanthroline-copper ion. Biopolymers. 1985 Jan;24(1):183–197. doi: 10.1002/bip.360240115. [DOI] [PubMed] [Google Scholar]
  25. Sigman D. S., Wahl G. M., Creighton D. J. Models for metalloenzymes. Zinc ion catalyzed phosphorylation of 1,10-phenanthroline-2-carbinol by adenosine triphosphate. Biochemistry. 1972 Jun 6;11(12):2236–2242. doi: 10.1021/bi00762a005. [DOI] [PubMed] [Google Scholar]
  26. Spassky A., Sigman D. S. Nuclease activity of 1,10-phenanthroline-copper ion. Conformational analysis and footprinting of the lac operon. Biochemistry. 1985 Dec 31;24(27):8050–8056. doi: 10.1021/bi00348a032. [DOI] [PubMed] [Google Scholar]
  27. Waring M. J. DNA modification and cancer. Annu Rev Biochem. 1981;50:159–192. doi: 10.1146/annurev.bi.50.070181.001111. [DOI] [PubMed] [Google Scholar]

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