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Nucleic Acids Research, 1980, Vol. 8, No. 12 2665-2678
© 1980

Articles

Analysis of DNA double- and single-strand breaks by two dimensional electrophorresis: action of micrococcal nuclease on chromatin and DNA, and degradation in vivo of lens fiber chromatin

Sohan P. Modak and Peter Beard

Swiss Institute for Experimental Cancer Research 1066 Epalinges, Switzerland

Received April 18, 1980. We describe a novel system for two dimensional electrophoresis at neutral and alkaline pH for determining the double-stranded and single-stranded lengths of DNA. With this system we analysed the mode of micrococcal nuclease digestion of DNA in cellular and SV4O viral chroniatin and of supercoiled SV40 DNA. The enzyme reaction occurred in two steps : the enzyme first introduced single-strand breaks, then converted these to double-strand breaks by an adjacent cleavage on the opposite strand. Digestion of cellular chromatin DNA occurred by a similar mechanism. Chromatin fragments produced by limited micrococcal nuclease action contained many single-strand breaks, which may be important when this method is used to prepare chromatin fragments for biochemical and biophysical studies. Nucleosome monomer to tetramer produced at later stages of digestion contained few if any single-strand breaks.


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