Methods for the detection of single-strand breaks in DNA under neutral conditions and their application in a study on the mechanism of repair of N-methylated purines in mouse cells.
Considering enzymatic activities found in bacteria and in animal cells, there are two possible mechanisms for repair of N-methylated purines produced by methylating agents such as the mutagen and carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Both mechanisms involve first an enzymatic removal of the methylated bases by glycosylases. The resulting apurinic sites could then be repaired by (a) direct insertion of the correct bases purine insertases or (b) opening of the polynucleotide chain by apurinic endonuclease followed by repair synthesis. As the methods commonly used to detect lesions induced by methylating agents involve alkali, it was thus far not possible to decide between the above possibilities because apurinic sites are by themselves alkali labile. In this paper I describe two methods which avoid alkali and therefore allow the clarification of some aspects of the repair reaction. One of these methods makes use of 95% formamide at 40 degrees C in place of alkali to denature DNA with pre-existing single-strand breaks, the other measures the capacity of DNA scissions with free 3'-OH groups to act as primer for Escherichia coli DNA polymerase I. Results obtained with both methods make it unlikely that purine insertases play a major role in the repair of apurinic sites. Kinetics of production and repair of single-strand breaks, produced in 3T6 mouse fibroblasts by incubation with N-methyl-N'-nitro-N-nitrosoguanidine, were also examined using the methods of alkaline elution and alkaline sucrose gradient centrifugation.
D000468:Alkalies; D000818:Animals; D004260:DNA Repair / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D005347:Fibroblasts / Q000378:metabolism; D066298:In Vitro Techniques; D008745:Methylation; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D051379:Mice; D009153:Mutagens / Q000494:pharmacology; D011687:Purines / Q000378:metabolism*
D000468:Alkalies; D004277:DNA, Single-Stranded; D009153:Mutagens; D011687:Purines; D008769:Methylnitronitrosoguanidine