Abstract
Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.
Highlights
Triple-stranded nucleic acids are complexes formed by three DNA or RNA complementary chains and may be composed of both RNA and DNA [1,2]
The labelling pattern obtained with antibodies to triplex nucleic acids was similar to that observed for the location of AAGAG and AAGAGAG repeats in mitotic chromosomes of observed for the location of AAGAG and AAGAGAG repeats in mitotic chromosomes of Drosophila
Sequences reactive with anti-triplex antibodies were identified in chromosomes larvae carrying the brownDominant allele, characterized by the AAGAG/AAGAGAG tandem from larvae carrying the brownDominant allele, characterized by the AAGAG/AAGAGAG tandem repeat block inserted into the polytene section 59E [23], which appeared strongly labelled by the repeat block inserted into the polytene section 59E [23], which appeared strongly labelled by the antibodies (Figure 4, Supplementary Figure S3)
Summary
Triple-stranded nucleic acids are complexes formed by three DNA or RNA complementary chains and may be composed of both RNA and DNA [1,2]. In addition to the characterization of proteins that probably act on triplex DNA in vivo, further evidence for triple-stranded DNA in chromosomes has been presented [18,19] but most data available in the literature remain still indirect since in vitro and in silico results, albeit important, do not prove complex formation in vivo. High resolution techniques such as NMR cannot be applied on chromatin or chromosomes. Tentative hypotheses on the functional involvement of three-stranded DNA structures in Drosophila heterochromatin are presented
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