Frequency Of Double-strand Breaks Research Articles

DNA double-strand breaks in mammalian cells after exposure to intercalating agents

Previous work has shown that exposing mouse L1210 cells to intercalating agents such as adriamycin, ellipticine and actinomycin D results in DNA single-strand breaks and DNA-protein crosslinks. To characterize further the interaction between these drugs and intracellular DNA we have employed a modification of the alkaline elution technique which allows the detection of DNA double-strand breaks. Ellipticine (1.25–5.0 μg/ml), adriamycin (0.5–3.0 μg/ml) and actinomycin D (1.5–3.0 μg/ml) all caused double-strand breaks in DNA from L1210 cells following a 1 h treatment. The number of double-strand breaks found per single strand break was highest for ellipticine, despite the fact that this is the least cytotoxic of the three drugs. By comparing the single and double strand break frequency caused by radiation to that caused by ellipticine, it appears that most if not all of the drug-induced single strand breaks observed actually represent double-strand breaks. We suggest that these double-strand breaks may result from the action of an intracellular enzyme, perhaps topoisomerase, which breaks both strands in concert to relieve the topological strain caused by drug intercalation.

A method for measuring the number of single- and double-strand breaks introduced into DNA molecules by the action of deoxyribonucleases

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 of 32P 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.

The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control.

With the use of neutral sucrose sedimentation techniques, the size of unirradiated nuclear DNA and the repair of double-strand breaks induced in it by ionizing radiation have been determined in both wild-type and homozygous rad52 diploids of the yeast Saccharomyces cerevisiae. The number average molecular weight of unirradiated DNA in these experiments is 3.0 X 10(8)+/-0.3 Daltons. Double-strand breaks are induced with a frequency of 0.58 X 10(-10) per Daltonkrad in the range of 25 to 100 krad. Since repair at low doses is observed in wild-type but not homozygous rad52 strains, the corresponding rad52 gene product is concluded to have a role in the repair process. Cycloheximide was also observed to inhibit repair to a limited extent indicating a requirement for protein synthesis. Based on the sensitivity of various mutants and the induction frequency of double-strand breaks, it is concluded that there are 1 to 2 double-strand breaks per lethal event in diploid cells incapable of repairing these breaks.