Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH

Human blood leukocytes within an agarose: matrix were deproteinized and exp osed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile s ites. Since the amount of ssDNA produced within a specific sequence area ma y be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FIS H). The DBD-FISH signal, corrected for the respective FISH signals in metap hase, was remarkably strong in the 5 bp classical satellite DNA domains ana lyzed (D1Z1, D9Z3. DYZ1), intermediate in the classical satellite 1 DNA seq uences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromer es). This result is evidence of a high density of constitutive alkali-labil e sites, probably abasic sites, within the 5 bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labil e sites could explain the high frequency of spontaneous breakage and rearra ngements in pericentromeric heterochromatin of chromosomes 1, 9. and 16, bu t not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment. (C) 20 01 Elsevier Science B.V. All rights reserved.