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. 1975 Aug;56(2):446–457. doi: 10.1172/JCI108111

Deoxyribonucleic acid strandedness. Partial characterization of the antigenic regions binding antibodies in lupus erythematosus serum.

R J Samaha, W S Irvin
PMCID: PMC436605  PMID: 125288

Abstract

This study shows that tritiated thymidine labeled DNA prepared from mammalian cells by the Marmur technique is a pure preparation of nucleic acid that is composed essentially of two populations of molecules. One molecular population consists of primarily double-standed nucleic acid, while the other population is of double-stranded nucleic acid with significant single-stranded regions. The double-stranded DNA with single-stranded regions can, depending upon the length of the single strand, behave as "native" DNA or "denatured" DNA on methylated albumin kieselguhr (MAK) column chromatography, Using MAK chromatography we have separated the DNA into a saltelutable fraction composed of primarily double-stranded molecules and an alkaline-elutable fraction containing double-stranded nucleic acid with variable length, single-stranded regions. Endonuclease enzyme removal of the single-stranded regions from the alkaline fraction DNA yield nucleic acid that behaves identically to the salt elutable DNA. Exonuclease removal of the single-stranded regions suggests they are located primarily at the ends of the molecules. Our data show that the alkaline-elutable DNA differs from salt-elutable DNA only in that the former has significant single-stranded regions. Sera of patients with systemic lupus erythematosus (SLE) selected for anti-DNA by hemagglutination bind significantly less to the alkaline fraction DNA than the sale fraction DNA. This difference in binding clearly does not represent simply an affinity for double-stranded vs. single-stranded nucleic acid since the alkaline fraction DNA contains predominately double-stranded nucleic acid. A model for antibody-DNA binding is suggested from the present data and information contained in the literature.

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Selected References

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

  1. Arana R., Seligmann M. Antibodies to native and denatured deoxyribonucleic acid in systemic lupus erythematosus. J Clin Invest. 1967 Nov;46(11):1867–1882. doi: 10.1172/JCI105677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BURGI E., HERSHEY A. D. Sedimentation rate as a measure of molecular weight of DNA. Biophys J. 1963 Jul;3:309–321. doi: 10.1016/s0006-3495(63)86823-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bernardi G. Chromatography of nucleic acids on hydroxyapatite. I. Chromatography of native DNA. Biochim Biophys Acta. 1969 Feb 18;174(2):423–434. doi: 10.1016/0005-2787(69)90273-1. [DOI] [PubMed] [Google Scholar]
  5. DAVISON P. F. Sedimentation of deoxyribonucleic acid isolated under low hydrodynamic shear. Nature. 1960 Mar 26;185:918–920. doi: 10.1038/185918a0. [DOI] [PubMed] [Google Scholar]
  6. DOTY P., MARMUR J., SUEOKA N. The heterogeneity in properties and functioning of deoxyribonucleic acids. Brookhaven Symp Biol. 1959 Nov;12:1–16. [PubMed] [Google Scholar]
  7. Epstein W. V., Tan M., Easterbrook M. Serum antibody to double-stranded RNA and DNA in patients with idiopathic and secondary uveitis. N Engl J Med. 1971 Dec 30;285(27):1502–1506. doi: 10.1056/NEJM197112302852703. [DOI] [PubMed] [Google Scholar]
  8. HERSHEY A. D., GOLDBERG E., BURGI E., INGRAHAM L. Local denaturation of DNA by shearing forces and by heat. J Mol Biol. 1963 Mar;6:230–243. doi: 10.1016/s0022-2836(63)80072-8. [DOI] [PubMed] [Google Scholar]
  9. Hasselbacher P., LeRoy E. C. Serum DNA binding activity in healthy subjects and in rheumatic disease. Arthritis Rheum. 1974 Jan-Feb;17(1):63–71. doi: 10.1002/art.1780170110. [DOI] [PubMed] [Google Scholar]
  10. Kaplan J. C., Kushner S. R., Grossman L. Enzymatic repair of DNA, 1. Purification of two enzymes involved in the excision of thymine dimers from ultraviolet-irradiated DNA. Proc Natl Acad Sci U S A. 1969 May;63(1):144–151. doi: 10.1073/pnas.63.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaplan J. C., Kushner S. R., Grossman L. Enzymatic repair of DNA. 3. Properties of the UV-endonuclease and UV-exonuclease. Biochemistry. 1971 Aug 31;10(18):3315–3324. doi: 10.1021/bi00794a001. [DOI] [PubMed] [Google Scholar]
  12. Koffler D., Carr R., Agnello V., Thoburn R., Kunkel H. G. Antibodies to polynucleotides in human sera: antigenic specificity and relation to disease. J Exp Med. 1971 Jul 1;134(1):294–312. doi: 10.1084/jem.134.1.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LACKS S. Molecular fate of DNA in genetic transformation of Pneumococcus. J Mol Biol. 1962 Jul;5:119–131. doi: 10.1016/s0022-2836(62)80067-9. [DOI] [PubMed] [Google Scholar]
  14. LINN S., LEHMAN I. R. AN ENDONUCLEASE FROM NEUROSPORA CRASSA SPECIFIC FOR POLYNUCLEOTIDES LACKING AN ORDERED STRUCTURE. I. PURIFICATION AND PROPERTIES OF THE ENZYME. J Biol Chem. 1965 Mar;240:1287–1293. [PubMed] [Google Scholar]
  15. LINN S., LEHMAN I. R. AN ENDONUCLEASE FROM NEUROSPORA CRASSA SPECIFIC FOR POLYNUCLEOTIDES LACKING AN ORDERED STRUCTURE. II. STUDIES OF ENZYME SPECIFICITY. J Biol Chem. 1965 Mar;240:1294–1304. [PubMed] [Google Scholar]
  16. 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]
  17. MANDELL J. D., HERSHEY A. D. A fractionating column for analysis of nucleic acids. Anal Biochem. 1960 Jun;1:66–77. doi: 10.1016/0003-2697(60)90020-8. [DOI] [PubMed] [Google Scholar]
  18. Pincus T., Schur P. H., Rose J. A., Decker J. L., Talal N. Measurement of serum DNA-binding activity in systemic lupus erythematosus. N Engl J Med. 1969 Sep 25;281(13):701–705. doi: 10.1056/NEJM196909252811304. [DOI] [PubMed] [Google Scholar]
  19. Pyeritz R. E., Schlegel R. A., Thomas C. A., Jr Hydrodynamic shear breakage of DNA may produce single-chained terminals. Biochim Biophys Acta. 1972 Jul 31;272(4):504–509. doi: 10.1016/0005-2787(72)90505-9. [DOI] [PubMed] [Google Scholar]
  20. Rabin E. Z., Fraser M. J. Isolation of Neurospora crassa endonuclease specific for single-stranded DNA. Can J Biochem. 1970 Mar;48(3):389–392. doi: 10.1139/o70-063. [DOI] [PubMed] [Google Scholar]
  21. Rabin E. Z., Mustard M., Fraser M. J. Specific inhibition by ATP and other properites of an endonuclease of Neurospora crassa. Can J Biochem. 1968 Oct;46(10):1285–1291. doi: 10.1139/o68-193. [DOI] [PubMed] [Google Scholar]
  22. Rabin E. Z., Preiss B., Fraser M. J. A nuclease from Neurospora crassa conidia specific for single-stranded nucleic acids. Prep Biochem. 1971;1(4):283–307. doi: 10.1080/00327487108081946. [DOI] [PubMed] [Google Scholar]
  23. Robitaille P., Tan E. M. Relationship between deoxyribonucleoprotein and deoxyribonucleic acid antibodies in systemic lupus erythematosus. J Clin Invest. 1973 Feb;52(2):316–323. doi: 10.1172/JCI107187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. SCHILDKRAUT C. L., MARMUR J., DOTY P. Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J Mol Biol. 1962 Jun;4:430–443. doi: 10.1016/s0022-2836(62)80100-4. [DOI] [PubMed] [Google Scholar]
  25. STOLLAR D., LEVINE L., LEHRER H. I., VAN VUNAKIS H. The antigenic determinants of denatured DNA reactive with lupus erythematosus serum. Proc Natl Acad Sci U S A. 1962 May 15;48:874–880. doi: 10.1073/pnas.48.5.874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. SUEOKA N., MARMUR J., DOTY P., 2nd Dependence of the density of deoxyribonucleic acids on guanine-cytosine content. Nature. 1959 May 23;183(4673):1429–1431. doi: 10.1038/1831429a0. [DOI] [PubMed] [Google Scholar]
  27. Schur P. H., Sandson J. Immunologic factors and clinical activity in systemic lupus erythematosus. N Engl J Med. 1968 Mar 7;278(10):533–538. doi: 10.1056/NEJM196803072781004. [DOI] [PubMed] [Google Scholar]
  28. Sharp G. C., Irvin W. S., LaRoque R. L., Velez C., Daly V., Kaiser A. D., Holman H. R. Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest. 1971 Feb;50(2):350–359. doi: 10.1172/JCI106502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tan E. M., Natali P. G. Comparative study of antibodies to native and denatured DNA. J Immunol. 1970 Apr;104(4):902–906. [PubMed] [Google Scholar]
  30. Tan E. M., Schur P. H., Carr R. I., Kunkel H. G. Deoxybonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J Clin Invest. 1966 Nov;45(11):1732–1740. doi: 10.1172/JCI105479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tan M., Epstein W. V. A solid-phase immunoassay for antibody to DNA and RNA. J Lab Clin Med. 1973 Jan;81(1):122–132. [PubMed] [Google Scholar]
  32. Wold R. T., Young F. E., Tan E. M., Farr R. S. Deoxyribonucleic acid antibody: a method to detect its primary interaction with deoxyribonucleic acid. Science. 1968 Aug 23;161(3843):806–807. doi: 10.1126/science.161.3843.806. [DOI] [PubMed] [Google Scholar]

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