We have shown previously that the neutral filter elution assay is dependent not only on the number of DNA double-strand breaks present in a mammalian cell but also on the way in which DNA expands on the filter following lysis. Results in this study indicate that the rate of DNA elution appears to be dependent upon the proximity of the DNA in relation to the replication complex. The rate of elution for DNA analyzed immediately after a 30-min labeling period with [14 C]thymidine was about five times slower than the rate of elution for bulk-labeled DNA. However, the rate was increased a few hours later when the recently replicated DNA had matured and was likely to be farther from replication-associated attachment sites on the nuclear protein matrix. About one cell cycle after pulse labeling, when the labeled DNA was replicated again, DNA underwent similar changes in elution rate. For the four cell lines examined here, the elution rate 3-4 h after pulse labeling correlated with cellular radiosensitivity. Changes in rate of elution caused by altering EDTA concentration or pH may also be explained by DNA structural changes which occur during lysis. We conclude that the neutral filter elution method is sensitive to differences in chromatin organization which may also play a role in cell sensitivity to ionizing radiation.

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