A method for measuring the number of single- and double-strand breaks introduced into DNA molecules by the action of deoxyribonucleases. |
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Authors: | A S Douvas |
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Institution: | Department of Medicine, Division of Rheumatic Diseases, University of Colorado Medical School, 4200 East Ninth Avenue, Denver, Colorado 80262 USA |
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Abstract: | A method is described for measuring the average number of nuclease-induced single- and double-strand breaks per DNA molecule. The procedure involves measuring the weight-average molecular weight of DNase I-digested DNA under neutral and alkaline conditions. A statistical equation is used to calculate the number of breaks per single- or double-stranded DNA molecule from the respective weight-average molecular weights. Enzymatic incorporation of32P into the 5′-OH ends of DNase I-induced breaks gave an independent measurement of the number of breaks per DNA molecule. Results obtained by the two different methods were in good agreement. In agreement with earlier reports we find that magnesium-activated DNase catalyzes a high frequency of single-strand breaks in DNA. The frequency of double-strand breaks is low, but significantly higher than can be explained by random accumulation of single-strand breaks. Our data suggest that the frequency of double-strand scission is affected by DNase-metal ion interactions. |
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