DNA double-strand breaks measured by the non-denaturing filter elution technique following X-ray and restriction endonuclease treatment
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The non-denaturing filter elution technique of Bradley and Kohn (1979) is widely used as an assay for DNA double-strand breaks (dsb) in mammalian cells. Results characteristically obtained with this assay following exposure of cells to ionising radiation, namely that of non-linear induction of dsb and rapid biphasic repair, are not in agreement with those of the neutral velocity sedimentation technique where the biophysical basis of measurement is understood. The discrepancy in the results obtained with these two techniques with regard to both Induction and repair of dsb has led to a controversy. In particular concerning the manner in which the data obtained with the neutral elution technique should be interpreted (AhnstrSm's Comment on Radford 1985; Hutchinson 1989). The aim of this project was therefore to attempt to test the assumed specificity of the non-denaturing filter elution technique as an assay for dsb. Optimization of the lysis and eluting conditions was followed by detailed X-ray dose-response and repair experiments with the CHO K1 cell line. A comparative study was performed using the xrs 5 cell line, a radiosensitive mutant of the CHO K1 line chosen for its characteristic marked deficiency in dsb repair, yet normal ability to rejoin single-strand breaks (Kemp et al 1984; Costa and Bryant 1988). Previous reports by Bryant and Blocher (1982), and Iliakis and Bryant (1983) revealed that the DNA synthesis Inhibitors ara A and ara C strongly inhibit dsb repair as assayed by neutral velocity sedimentation. I thus adopted an experimental strategy In which the effect of ara A and ara C on putative dsb repair was examined using the non- denaturing filter elution assay. Only limited inhibition of dsb repair by these nucleoside analogues was observed with the non-denaturing filter elution technique tn contrast to the complete inhibition of dsb repair as measured by neutral velocity sedimentation for the same concentrations of DNA synthesis inhibitor (Bryant and Blocher 1982; Iliakis and Bryant 1983). These results suggest that the two above mentioned techniques are detecting disparate types of dsb, as manifested by the differential requirement of the repair mechanism of these breaks for DNA polymerization. A further approach was the introduction of restriction endonucleases (RE) into mammalian cells by electroporation, to induce dsb in the absence of other types of lesions. The observed increase in the rate of elution of the DNA of RE-treated cells substantiates the ability of the non-denaturing filter elution assay to detect cellular dsb. Surprisingly Pvu II was found to remain active inside the cell for up to 24 h, and the continual incision of the DNA by the enzyme thwarted the possibility of monitoring the repair of these dsb. A noteworthy result was the relative inability of RE which generate cohesive-ended dsb (e.g. Bam HI and Eco R1) to Induced measurable numbers of dsb as compared with the blunt-end cutter Pvu II. A hypothesis of a competition between the induction of dsb by RE and the subsequent repair is offered as explanation, where the repair of cohesive-ended dsb is assumed to take place at a higher rate than that of blunt-ended dsb. A comparative study using the xrs 5 mutant cell line, known to be deficient in dsb repair, revealed enhanced levels of RE-induced dsb which would support the notion that the levels of dsb reflect a competition between RE-incision and dsb repair. In summary, this study validates the measurement of dsb by the non-denaturing filter elution method and provides new evidence for the mode of induction of dsb by RE which has not been hitherto possible. Finally, the work indicates the way in which cells handle different types of dsb which may be similar to the manner In which the variety of dsb induced by ionising radiation are dealt with.
Thesis, PhD Doctor of Philosophy
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