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. 2000 Nov;79(5):2692–2704. doi: 10.1016/S0006-3495(00)76507-6

Multimerization-cyclization of DNA fragments as a method of conformational analysis.

A A Podtelezhnikov 1, C Mao 1, N C Seeman 1, A Vologodskii 1
PMCID: PMC1301149  PMID: 11053141

Abstract

Ligation of short DNA fragments results in the formation of linear and circular multimers of various lengths. The distribution of products in such a reaction is often used to evaluate fragment bending caused by specific chemical modification, by bound ligands or by the presence of irregular structural elements. We have developed a more rigorous quantitative approach to the analysis of such experimental data based on determination of j-factors for different multimers from the distribution of the reaction products. j-Factors define the effective concentration of one end of a linear chain in the vicinity of the other end. To extract j-factors we assumed that kinetics of the reaction is described by a system of differential equations where j-factors appear as coefficients. The assumption was confirmed by comparison with experimental data obtained here for DNA fragments containing A-tracts. At the second step of the analysis j-factors are used to determine conformational parameters of DNA fragments: the equilibrium bend angle, the bending rigidity of the fragment axis, and the total twist of the fragments. This procedure is based on empirical equations that connect the conformational parameters with the set of j-factors. To obtain the equations, we computed j-factors for a large array of conformational parameters that describe model fragments. The approach was tested on both simulated and actual experimental data for DNA fragments containing A-tracts. A-tract DNA bend angle determined here is in good agreement with previously published data. We have established a set of experimental conditions necessary for the data analysis to be successful.

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Selected References

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  1. Balagurumoorthy P., Sakamoto H., Lewis M. S., Zambrano N., Clore G. M., Gronenborn A. M., Appella E., Harrington R. E. Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8591–8595. doi: 10.1073/pnas.92.19.8591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caruthers M. H. Gene synthesis machines: DNA chemistry and its uses. Science. 1985 Oct 18;230(4723):281–285. doi: 10.1126/science.3863253. [DOI] [PubMed] [Google Scholar]
  3. Crothers D. M., Drak J., Kahn J. D., Levene S. D. DNA bending, flexibility, and helical repeat by cyclization kinetics. Methods Enzymol. 1992;212:3–29. doi: 10.1016/0076-6879(92)12003-9. [DOI] [PubMed] [Google Scholar]
  4. Crothers D. M., Haran T. E., Nadeau J. G. Intrinsically bent DNA. J Biol Chem. 1990 May 5;265(13):7093–7096. [PubMed] [Google Scholar]
  5. Dlakic M., Harrington R. E. Bending and torsional flexibility of G/C-rich sequences as determined by cyclization assays. J Biol Chem. 1995 Dec 15;270(50):29945–29952. doi: 10.1074/jbc.270.50.29945. [DOI] [PubMed] [Google Scholar]
  6. Dlakić M., Harrington R. E. The effects of sequence context on DNA curvature. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):3847–3852. doi: 10.1073/pnas.93.9.3847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hagerman P. J., Ramadevi V. A. Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure. I. Computational analysis. J Mol Biol. 1990 Mar 20;212(2):351–362. doi: 10.1016/0022-2836(90)90130-E. [DOI] [PubMed] [Google Scholar]
  8. Kahn J. D., Crothers D. M. Measurement of the DNA bend angle induced by the catabolite activator protein using Monte Carlo simulation of cyclization kinetics. J Mol Biol. 1998 Feb 13;276(1):287–309. doi: 10.1006/jmbi.1997.1515. [DOI] [PubMed] [Google Scholar]
  9. Kahn J. D., Crothers D. M. Protein-induced bending and DNA cyclization. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6343–6347. doi: 10.1073/pnas.89.14.6343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kerppola T. K. Fos and Jun bend the AP-1 site: effects of probe geometry on the detection of protein-induced DNA bending. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10117–10122. doi: 10.1073/pnas.93.19.10117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Klenin K. V., Vologodskii A. V., Anshelevich V. V., Dykhne A. M., Frank-Kamenetskii M. D. Computer simulation of DNA supercoiling. J Mol Biol. 1991 Feb 5;217(3):413–419. doi: 10.1016/0022-2836(91)90745-r. [DOI] [PubMed] [Google Scholar]
  12. Koo H. S., Drak J., Rice J. A., Crothers D. M. Determination of the extent of DNA bending by an adenine-thymine tract. Biochemistry. 1990 May 1;29(17):4227–4234. doi: 10.1021/bi00469a027. [DOI] [PubMed] [Google Scholar]
  13. Levene S. D., Crothers D. M. Ring closure probabilities for DNA fragments by Monte Carlo simulation. J Mol Biol. 1986 May 5;189(1):61–72. doi: 10.1016/0022-2836(86)90381-5. [DOI] [PubMed] [Google Scholar]
  14. Lyubchenko Y. L., Shlyakhtenko L. S., Appella E., Harrington R. E. CA runs increase DNA flexibility in the complex of lambda Cro protein with the OR3 site. Biochemistry. 1993 Apr 20;32(15):4121–4127. doi: 10.1021/bi00066a038. [DOI] [PubMed] [Google Scholar]
  15. Lyubchenko Y., Shlyakhtenko L., Chernov B., Harrington R. E. DNA bending induced by Cro protein binding as demonstrated by gel electrophoresis. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5331–5334. doi: 10.1073/pnas.88.12.5331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nagaich A. K., Zhurkin V. B., Sakamoto H., Gorin A. A., Clore G. M., Gronenborn A. M., Appella E., Harrington R. E. Architectural accommodation in the complex of four p53 DNA binding domain peptides with the p21/waf1/cip1 DNA response element. J Biol Chem. 1997 Jun 6;272(23):14830–14841. doi: 10.1074/jbc.272.23.14830. [DOI] [PubMed] [Google Scholar]
  17. Rivetti C., Walker C., Bustamante C. Polymer chain statistics and conformational analysis of DNA molecules with bends or sections of different flexibility. J Mol Biol. 1998 Jul 3;280(1):41–59. doi: 10.1006/jmbi.1998.1830. [DOI] [PubMed] [Google Scholar]
  18. Shlyakhtenko L. S., Appella E., Harrington R. E., Kutyavin I., Lyubchenko Y. L. Structure of three-way DNA junctions. 2. Effects of extra bases and mismatches. J Biomol Struct Dyn. 1994 Aug;12(1):131–143. doi: 10.1080/07391102.1994.10508092. [DOI] [PubMed] [Google Scholar]
  19. Shore D., Baldwin R. L. Energetics of DNA twisting. I. Relation between twist and cyclization probability. J Mol Biol. 1983 Nov 15;170(4):957–981. doi: 10.1016/s0022-2836(83)80198-3. [DOI] [PubMed] [Google Scholar]
  20. Shore D., Langowski J., Baldwin R. L. DNA flexibility studied by covalent closure of short fragments into circles. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4833–4837. doi: 10.1073/pnas.78.8.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Taylor W. H., Hagerman P. J. Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure. II. NaCl-dependence of DNA flexibility and helical repeat. J Mol Biol. 1990 Mar 20;212(2):363–376. doi: 10.1016/0022-2836(90)90131-5. [DOI] [PubMed] [Google Scholar]
  22. Ulanovsky L., Bodner M., Trifonov E. N., Choder M. Curved DNA: design, synthesis, and circularization. Proc Natl Acad Sci U S A. 1986 Feb;83(4):862–866. doi: 10.1073/pnas.83.4.862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang J. C. Cyclization of coliphage 186 DNA. J Mol Biol. 1967 Sep 28;28(3):403–411. doi: 10.1016/s0022-2836(67)80089-5. [DOI] [PubMed] [Google Scholar]
  24. Wang J. C., Davidson N. Cyclization of phage DNAs. Cold Spring Harb Symp Quant Biol. 1968;33:409–415. doi: 10.1101/sqb.1968.033.01.047. [DOI] [PubMed] [Google Scholar]
  25. Wang J. C., Davidson N. On the probability of ring closure of lambda DNA. J Mol Biol. 1966 Aug;19(2):469–482. doi: 10.1016/s0022-2836(66)80017-7. [DOI] [PubMed] [Google Scholar]
  26. Wang J. C., Davidson N. Thermodynamic and kinetic studies on the interconversion between the linear and circular forms of phage lambda DNA. J Mol Biol. 1966 Jan;15(1):111–123. doi: 10.1016/s0022-2836(66)80213-9. [DOI] [PubMed] [Google Scholar]
  27. Wang J. C. Helical repeat of DNA in solution. Proc Natl Acad Sci U S A. 1979 Jan;76(1):200–203. doi: 10.1073/pnas.76.1.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Xu R., Mao B., Amin S., Geacintov N. E. Bending and circularization of site-specific and stereoisomeric carcinogen-DNA adducts. Biochemistry. 1998 Jan 13;37(2):769–778. doi: 10.1021/bi971785x. [DOI] [PubMed] [Google Scholar]
  29. Zeman S. M., Depew K. M., Danishefsky S. J., Crothers D. M. Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand-DNA complexes: the interaction between DNA and calichearubicin B. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4327–4332. doi: 10.1073/pnas.95.8.4327. [DOI] [PMC free article] [PubMed] [Google Scholar]

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