Abstract
At various stages of the life cycle of cells, the occurrence of DNA single-strand breaks (SSB) is a common event. In order to understand better the relationship of SSB to physiological states of cells, one requires the analysis of SSB at the level of individual cells by in situ nick translation (ISNT). In the principle of ISNT, the DNA strand with breaks is elongated in the presence of biotin-11-dUTP by E. coil DNA polymerase I at the nicked sites. The biotin moieties incorporated into the newly synthesized strand are visualized enzyme-immunohistochemically with horseradish peroxidase-labeled antibiotin antibody. In this article, I will firstly describe the details of optimization of the ISNT reaction for its full implementation, using nicked λ phage DNA, which was fixed onto nitrocellulose filters, as well as using fresh frozen sections of rat testis and small intestine. Subsequently, I would like to demonstrate the presence of two types of SSB, which can be practically distinguished by protease-dependency of the staining; one is readily detected by ISNT without any deproteination steps (protease-independent type), and the other requires the protease treatment to be detected by ISNT (protease-dependent type). In our case, the single-strand breaks of DNA observed in terminally differentiated cells as well as the cells undergoing necrosis were protease-independent type. On the other hand, the DNA breaks in the cells undergoing replicative DNA synthesis and apoptosis were regarded as the latter type. Consequently, ISNT should be regarded as a useful molecular histochemical tool to categorize naturally occurring SSB.
