Open AccessInteraction of DAPI with individual strands of trinucleotide repeats
Author(s) -
Trotta Edoardo,
Del Grosso Nicoletta,
Erba Maura,
Melino Sonia,
Cicero Daniel,
Paci Maurizio
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03877.x
Subject(s) - dapi , dna , biology , trinucleotide repeat expansion , biophysics , microbiology and biotechnology , chemistry , genetics , staining , gene , allele
The structural changes produced by the minor‐groove binding ligand DAPI (4′,6‐diamidine‐2‐phenylindole) on individual strands of trinucleotide repeat sequences were detected by electrophoretic band‐shift analysis and related to their effects on DNA replication in vitro . Among the 20 possible single‐stranded trinucleotide repeats, only the T‐rich strand of the AAT·ATT triplet exhibits an observable fluorescence band and a change in electrophoretic mobility due to the drug binding. This is attributable to the property of DAPI that favours folding of the random coil ATT strand into a fast‐migrating hairpin structure by a minor‐groove binding mechanism. Electrophoretic characteristics of AAT, ACT, AGT, ATG and ATC are unchanged by DAPI, suggesting the crucial role of T·T with respect to A·A, C·C and G·G mismatch, in favouring the binding properties and the structural features of the ATT–DAPI complexes. Primer extension experiments, using the Klenow fragment of DNA polymerase I, demonstrate that such a selective structural change at ATT targets presents a marked property to stall DNA replication in vitro in comparison with the complementary AAT and a random GC‐rich sequence. The results suggest a novel molecular mechanism of action of the DNA minor‐groove binding ligand DAPI.