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 exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.
Fernández
J L
JL
Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, 15009, La Coruña, Spain. genetica@cog.es
Vázquez-Gundín
F
F
Rivero
M T
MT
Goyanes
V
V
Gosálvez
J
J
D002503:Centromere; D019457:Chromosome Breakage; D004276:DNA, Satellite / Q000097:blood* / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000097:blood* / Q000737:chemistry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D066298:In Vitro Techniques; D007962:Leukocytes / Q000648:ultrastructure; D008297:Male; D009691:Nucleic Acid Denaturation; D012972:Sodium Hydroxide / Q000494:pharmacology*
D004276:DNA, Satellite; D004277:DNA, Single-Stranded; D012972:Sodium Hydroxide