ABSTRACT DNA damage and repair may be studied in situ in single cells at the level of the whole genome and/or within specific DNA sequence areas by the use of the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. In this technique, microgel-embedded cells are deproteinized and exposed to an unwinding solution that transforms DNA breaks into single-stranded DNA motifs, which may then be detected by a fluorescent DNA probe. The hybridization yield is proportional to the DNA breakage level, the DNA probe defining the specific chromatin area to be assessed. This has been initially applied to analyze the intragenomic heterogeneity in DNA damage induced by ionizing radiation in various human satellite DNA sequence areas from lymphocytes. Recently, it has become possible to discriminate ionizing radiation-induced DNA single- and double-strand breaks by intercalating a neutral electrophoresis step before the unwinding treatment. This technical variant of DBD-FISH has been used to assess DNA damage and repair in the extended blocks of interstitial telomeric repeat sequences from Chinese hamster chromosomes. This showed a lower rejoining rate of ionizing radiation-induced DNA double-strand breaks within interstitial telomeric repeat sequences, compared to that of the whole genome. The influence of the activity of specific DNA-repair proteins and Wortmannin, an inhibitor of DNA double-strand break repair, was also assessed within these specific targets, demonstrating intragenomic heterogeneity in DNA-damage induction and repair. This was particularly evident after exposure to nitric oxide donors. Moreover, besides DNA damage and repair, the DBD-FISH procedure allowed the study of constitutive modifications of the native DNA organization, specifically the relative distribution of alkali-labile sites among different sequence areas and in various cell types. Thus, interstitial telomeric repeat sequences, and particularly the whole genome of mature sperm cells, were found to be enriched in these DNA modifications. Finally, the DBD-FISH procedure applied to human sperm cells has led to the development of the Sperm Chromatin Dispersion (SCD) test for determining the presence of DNA fragmentation in this particular cell type. This test is being developed as a kit (Halosperm®), with separate variants for different species, to make possible the assessment of this new aspect of sperm quality in every basic laboratory. Overall, DBD-FISH is a technically flexible procedure that allows modifications to be adapted for a wide range of purposes.
Buy this Article
|
