Biopolym. Cell. 2012; 28(4):292-297.
http://dx.doi.org/10.7124/bc.000062
Structure and Function of Biopolymers
Influence of chloroquine on kinetics of single-cell gel electrophoresis
1Zazhytska M. O., 1Afanasieva K. S., 1Chopei M. I., 1Vikhreva M. A., 1Sivolob A. V.
  1. Educational and Scientific Center "Institute of Biology",
    Taras Shevchenko National University of Kyiv
    64/13, Volodymyrska Str., Kyiv, Ukraine, 01601

Abstract

In single-cell gel electrophoresis (the comet assay) the DNA of lysed cells, the nucleoids, extends towards the anode in a track resembling a comet tail. The aim of this work was to investigate the effects of changes in DNA topology on this process. Methods. We used the kinetic approach, proposed earlier by us, to measure a relative amount of DNA in the comet tails as a function of time in the presence of different concentrations of chloroquine, a widely used intercalator. Results. We have shown that, at given small concentrations, intercalation of chloroquine strongly facilitates the comet tail formation. At the same time, some part of DNA (about 8 %) in the nucleoids exits very fast independently on chloroquine, while the largest part of DNA (about three quarters) does not exit at all. At high concentrations the intercalator increases the fraction of DNA, which cannot exit. Conclusions. Our results imply that the loop domains, which contain about one to several hundreds kilobases, represent only a small part (about a quarter) of DNA in the nucleus. The intercalation induces detachment of these loops from the nuclear matrix.
Keywords: comet assay, chloroquine, intercalation, DNA loops, supercoiling
Full text: (PDF, in English)

References

[1] Olive P. L. The comet assay. An overview of techniques Methods Mol. Biol 2002 203:179–194.
[2] Collins A. R. The comet assay for DNA damage and repair: principles, applications, and limitations Mol. Biotechnol 2004 26, N 3:249–261.
[3] Dusinska M., Collins A. R. The comet assay in human biomonitoring: gene – environment interactions Mutagenesis 2008 23, N 3:191–205.
[4] Collins A. R., Oscoz A. A., Brunborg G., Gaivao I., Giovannelli L., Kruszewski M., Smith C. C., Stetina R. The comet assay: topical issues Mutagenesis 2008 23, N 3:143–151.
[5] Cook P. R., Brazell I. A., Jost E. Characterization of nuclear structures containing superhelical DNA J. Cell Sci 1976 22, N 2:303–324.
[6] Shaposhnikov S. A., Salenko V. B., Brunborg G., Nygren J., Collins A. R. Single-cell gel electrophoresis (the comet assay): loops or fragments? Electrophoresis 2008 29, N 14:3005– 3012.
[7] Afanasieva K. S., Shuvalova T. A., Zazhytska M. O., Sivolob A. V. Reversibility of DNA loops exit during single cell gel electrophoresis Biopolym. Cell 2008 24, N 2:105–111.
[8] Afanasieva K., Zazhytska M., Sivolob A. Kinetics of comet formation in single-cell gel electrophoresis: loops and fragments Electrophoresis 2010 31, N 3:512–519.
[9] Olive P. L., Banath J. P. The comet assay: a method to measure DNA damage in individual cells Nat. Protoc 2006 1, N 1:23–29.
[10] Cohen S. N., Yielding K. L. Spectrophotometric studies of the interaction of chloroquine with deoxyribonucleic acid J. Biol. Chem 1965 240, N 7:3123–3131.
[11] McGhee JD, von Hippel PH. Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. J Mol Biol. 1974;86(2):469-89.
[12] Sivolob A., De Lucia F., Revet B., Prunell A. Nucleosome dynamics II. High flexibility of nucleosome entering and exiting DNAs to positive crossing. An ethidium bromide fluorescence study of mononucleosomes on DNA minicircles J. Mol. Biol 1999 285, N 3:1081–1099.
[13] Bauer W., Vinograd J. Interaction of closed circular DNA with intercalative dyes II. The free energy of superhelix formation in SV40 DNA J. Mol. Biol 1970 47, N 3:419–435.
[14] Jones R. L., Lanier A. C., Keel R. A., Wilson W. D. The effect of ionic strength on DNA-ligand unwinding angles for acridine and quinoline derivatives Nucleic Acids Res 1980 8, N 7:1613–1624.
[15] Reese H. R. Effects of DNA charge and length on the electrophoretic mobility of intercalated DNA Biopolymers 1994 34, N 10:1349–1358.
[16] Sigmon J., Larcom L. L. The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis Electrophoresis 1996 17, N 10:1524–1527.
[17] Hilger I., Rapp A., Greulich K. O., Kaiser W. A. Assessment of DNA damage in target tumor cells after thermoablation in mice Radiobiology 2005 237, N 2:500–506.
[18] Sullivan R., Graham C. H. Hypoxia prevents etoposide-induced DNA damage in cancer cells through a mechanism involving hypoxia-inducible factor 1 Mol. Cancer Ther 2009 8, N 6:1702–1713.
[19] Barker G. F., Manzo N. D., Cotich K. L., Shone R. K., Waxman A. B. DNA damage induced by hyperoxia: quantitation and correlation with lung injury Am. J. Respir. Cell Mol. Biol 2006 35, N 3:277–288.
[20] Afanas'eva K. S., Zazhytskaia M. O., Sivolob A. V. Mechanisms of DNA exit during neutral and alkaline comet assay Tsitol. Genet 2009 43, N 6:3–7.