Immunoassays for carbodiimide modified DNA-detection of unpairing transitions in supercoiled ColE1 DNA (original) (raw)

Abstract

The water soluble reagent N-cyclohexyl-N'-beta-(4-methylmorpholinium) ethyl carbodiimide-p-toluene sulphonate (CMC) can be used to probe for unpaired and mismatched sites in DNA. Polyclonal antibodies for CMC modified DNA were produced in order to develop immunological assays for the localization and quantitation of CMC adducts. Immunoslot blot analysis of modified DNA exhibited antibody binding proportional to the extent of CMC modification with adduct detection in the femtamole range. Unmodified DNA did not cross react under the conditions of the assay. The distribution of CMC reactivity for supercoiled ColE1 DNA modified at 100, 200 and 300 mM NaCl was determined by immunoanalysis of EcoRI-Hae2-NruI restriction fragments Southern transferred to nylon membranes. Reactivity above random expectation occurred in the A2-II fragment which can be accounted for by its high A-T content of 71.3%. Reactivity below random expectation occurred in the C fragment which can be accounted for by its low AT content of 43%. CMC modification for the other restriction fragments appeared random.

9957

Images in this article

Selected References

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

  1. Beerman T. A., Lebowitz J. Further analysis of the altered secondary structure of superhelical DNA. Sensitivity to methylmercuric hydroxide a chemical probe for unpaired bases. J Mol Biol. 1973 Sep 25;79(3):451–470. doi: 10.1016/0022-2836(73)90398-7. [DOI] [PubMed] [Google Scholar]
  2. Benham C. J. Theoretical analysis of competitive conformational transitions in torsionally stressed DNA. J Mol Biol. 1981 Jul 25;150(1):43–68. doi: 10.1016/0022-2836(81)90324-7. [DOI] [PubMed] [Google Scholar]
  3. Bustin M. Immunological approaches to chromatin and chromosome structure and function. Curr Top Microbiol Immunol. 1979;88:105–142. doi: 10.1007/978-3-642-67331-3_3. [DOI] [PubMed] [Google Scholar]
  4. Cesarone C. F., Bolognesi C., Santi L. Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Anal Biochem. 1979 Nov 15;100(1):188–197. doi: 10.1016/0003-2697(79)90131-3. [DOI] [PubMed] [Google Scholar]
  5. Chan P. T., Ohmori H., Tomizawa J., Lebowitz J. Nucleotide sequence and gene organization of ColE1 DNA. J Biol Chem. 1985 Jul 25;260(15):8925–8935. [PubMed] [Google Scholar]
  6. Chen M., Lebowitz J., Salzman N. P. Hin D restriction mapping of upaired regions in simian virus 40 superhelical DNA I: considerations regarding structure-function relationships. J Virol. 1976 Apr;18(1):211–217. doi: 10.1128/jvi.18.1.211-217.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dean W. W., Lebowitz J. Partial alteration of secondary structure in native superhelical DNA. Nat New Biol. 1971 May 5;231(18):5–8. [PubMed] [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ehresmann C., Baudin F., Mougel M., Romby P., Ebel J. P., Ehresmann B. Probing the structure of RNAs in solution. Nucleic Acids Res. 1987 Nov 25;15(22):9109–9128. doi: 10.1093/nar/15.22.9109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grossman L., Caron P. R., Mazur S. J., Oh E. Y. Repair of DNA-containing pyrimidine dimers. FASEB J. 1988 Aug;2(11):2696–2701. doi: 10.1096/fasebj.2.11.3294078. [DOI] [PubMed] [Google Scholar]
  11. Hale P., Woodward R. S., Lebowitz J. Carbodiimide inactivation of Escherichia coli RNA polymerase promoters on supercoiled simian virus 40 and ColE1 DNAs occurs by a one-hit process at salt concentrations in the physiological range. J Biol Chem. 1983 Jun 25;258(12):7828–7839. [PubMed] [Google Scholar]
  12. Hale P., Woodward R. S., Lebowitz J. E. coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification. Nature. 1980 Apr 17;284(5757):640–644. doi: 10.1038/284640a0. [DOI] [PubMed] [Google Scholar]
  13. Kohwi Y., Kohwi-Shigematsu T. Magnesium ion-dependent triple-helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid DNA. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3781–3785. doi: 10.1073/pnas.85.11.3781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kowalski D., Natale D. A., Eddy M. J. Stable DNA unwinding, not "breathing," accounts for single-strand-specific nuclease hypersensitivity of specific A+T-rich sequences. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9464–9468. doi: 10.1073/pnas.85.24.9464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Leary J. J., Brigati D. J., Ward D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4045–4049. doi: 10.1073/pnas.80.13.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lebowitz J., Chaudhuri A. K., Gonenne A., Kitos G. Carbodiimide modification of superhelical PM2 DNA: considerations regarding reaction at unpaired bases and the unwinding of superhelical DNA with chemical probes. Nucleic Acids Res. 1977 Jun;4(6):1695–1711. doi: 10.1093/nar/4.6.1695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lebowitz J., Garon C. G., Chen M. C., Salzman N. P. Chemical modification of simian virus 40 DNA by reaction with a water-soluble carbodiimide. J Virol. 1976 Apr;18(1):205–210. doi: 10.1128/jvi.18.1.205-210.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Metz D. H., Brown G. L. The investigation of nucleic acid secondary structure by means of chemical modification with a carbodiimide reagent. I. The reaction between N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide and model nucleotides. Biochemistry. 1969 Jun;8(6):2312–2328. doi: 10.1021/bi00834a012. [DOI] [PubMed] [Google Scholar]
  19. Montandon A. J., Green P. M., Giannelli F., Bentley D. R. Direct detection of point mutations by mismatch analysis: application to haemophilia B. Nucleic Acids Res. 1989 May 11;17(9):3347–3358. doi: 10.1093/nar/17.9.3347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Novack D. F., Casna N. J., Fischer S. G., Ford J. P. Detection of single base-pair mismatches in DNA by chemical modification followed by electrophoresis in 15% polyacrylamide gel. Proc Natl Acad Sci U S A. 1986 Feb;83(3):586–590. doi: 10.1073/pnas.83.3.586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Palecek E., Krejcová A., Vojtísková M., Podgorodnichenko V., Ilyina T., Poverennyi A. Antibodies to DNAs chemically modified with osmium structural probes. Gen Physiol Biophys. 1989 Oct;8(5):491–504. [PubMed] [Google Scholar]
  22. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  24. Sullivan K. M., Murchie A. I., Lilley D. M. Long range structural communication between sequences in supercoiled DNA. Sequence dependence of contextual influence on cruciform extrusion mechanism. J Biol Chem. 1988 Sep 15;263(26):13074–13082. [PubMed] [Google Scholar]
  25. Thomas D. C., Kunkel T. A., Casna N. J., Ford J. P., Sancar A. Activities and incision patterns of ABC excinuclease on modified DNA containing single-base mismatches and extrahelical bases. J Biol Chem. 1986 Nov 5;261(31):14496–14505. [PubMed] [Google Scholar]
  26. Wani A. A., D'Ambrosio S. M., Alvi N. K. Quantitation of pyrimidine dimers by immunoslot blot following sublethal UV-irradiation of human cells. Photochem Photobiol. 1987 Oct;46(4):477–482. doi: 10.1111/j.1751-1097.1987.tb04798.x. [DOI] [PubMed] [Google Scholar]
  27. Wani A. A., D'Ambrosio S. M. Immunological quantitation of O4-ethylthymidine in alkylated DNA: repair of minor miscoding base in human cells. Carcinogenesis. 1987 Aug;8(8):1137–1144. doi: 10.1093/carcin/8.8.1137. [DOI] [PubMed] [Google Scholar]
  28. Wani A. A., Gibson-D'Ambrosio R. E., D'Ambrosio S. M. Antibodies to UV irradiated DNA: the monitoring of DNA damage by ELISA and indirect immunofluorescence. Photochem Photobiol. 1984 Oct;40(4):465–471. doi: 10.1111/j.1751-1097.1984.tb04619.x. [DOI] [PubMed] [Google Scholar]
  29. Wani A. A., Gibson-D'Ambrosio R. E., D'Ambrosio S. M. Quantitation of O6-ethyldeoxyguanosine in ENU alkylated DNA by polyclonal and monoclonal antibodies. Carcinogenesis. 1984 Sep;5(9):1145–1150. doi: 10.1093/carcin/5.9.1145. [DOI] [PubMed] [Google Scholar]
  30. Wani A. A., Hart R. W. Pisum sativum endonuclease. Studies on substrate specificity and possible use as a biochemical tool. Biochim Biophys Acta. 1981 Oct 27;655(3):396–406. doi: 10.1016/0005-2787(81)90051-4. [DOI] [PubMed] [Google Scholar]
  31. Wani A. A., Stephens R. E., D'Ambrosio S. M., Hart R. W. A sequence specific endonuclease from Micrococcus radiodurans. Biochim Biophys Acta. 1982 May 31;697(2):178–184. doi: 10.1016/0167-4781(82)90075-6. [DOI] [PubMed] [Google Scholar]
  32. Wells R. D., Collier D. A., Hanvey J. C., Shimizu M., Wohlrab F. The chemistry and biology of unusual DNA structures adopted by oligopurine.oligopyrimidine sequences. FASEB J. 1988 Nov;2(14):2939–2949. [PubMed] [Google Scholar]
  33. Wells R. D. Unusual DNA structures. J Biol Chem. 1988 Jan 25;263(3):1095–1098. [PubMed] [Google Scholar]