Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay.
In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.
Johnston
P J
PJ
Department of Biology and Pre-clinical medicine, University of St. Andrews, Fife, UK.
Bryant
P E
PE
D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D005374:Filtration; D006863:Hydrogen-Ion Concentration
D004277:DNA, Single-Stranded; D005047:Etoposide; D004247:DNA; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D004250:DNA Topoisomerases, Type II; D002166:Camptothecin