Photofootprinting of DNA triplexes

Abstract

We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6-4) pyrimidine photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined conditions. At acid pH, d(C)n.d(G)n.d(C)n and d(CT)n.d(GA)n.d(CT)n triplexes are detected by this method. The d(CT)n.d(GA)n.d(CT)n triplexes are additionally stabilized by divalent cations and spermidine. PyPuPu triplexes are pH-independent and are stabilized by divalent cations, such as Mg++ and Zn++. The effect depends on the type of cation and on the DNA sequence. The d(CT)n.d(GA)n.d(GA)n triplex is stabilized by Zn++, but not by Mg++, whereas the d(C)n.d(G)n.d(G)n triplex is stabilized by Mg++. In H-DNA, virtually the entire pyrimidine chain is protected against photodimerization, whereas only half of the pyrimidine chain participating in a triplex is protected in the CGG intramolecular triplex.

Photochemical probing of the B–A conformational transition in a linearized pUC19 DNA and its polylinker region

“…Success in these situations has partly been reported only with the use of UV light, which damages DNA, whereas the damage is sensitive to the physical state of DNA in the irradiated sample. The damage reflects contacts between the lac repressor and the operator in E. coli cells [47], regulatory protein-DNA interactions in mammalian genomic DNA [39], histone-DNA interaction [38], doublestranded and single-stranded states of the 5S rRNA gene [48], DNA arrangements into nucleosome cores [49], unusual conformation of (dA) n ·(dT) n tracts in DNA [50], transcription factor binding to DNA [51], EcoRI endonuclease binding to DNA [52], and DNA triplex formation [53][54][55][56].…”

The Interaction of Zinc(II) Ions with Antiparallel-Stranded d(GA)nDNA Homoduplexes

“…It was proposed that they would act through a dual mechanism, by destabilizing the double-stranded B-DNA conformation and stabilizing the [GA(GA-TC)] and (GA-GA) intramolecular forms (7). Consistent with this hypothesis, zinc is known to promote the formation of [GA(GA-TC)] intermolecular triplexes (8,9). It is also known that, in the presence of zinc, single-stranded d(GA)n DNA sequences can form two distinct types of homoduplexes one of which was proposed to be antiparallelstranded (10).…”

“…At present, it is uncertain whether zinc ions play a specific role on the stabilization of the [GA(GATC)] triplexes. On one hand, intramolecular [GA(GATC)] triplexes are destabilised at high zinc concentration giving rise to the formation of intramolecular (GAGA) hairpins (4,7) but, on the other hand, intermolecular [GA(GATC)] triplexes are stabilized by zinc (8,9). From our results, the specific interaction of zinc with anti parallel-stranded d(GA)n DNA appears as a main event driving the structural transitions that zinc induces at the d(GATC)n sequences.…”

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn2+ ions

“…For instance Mg 2+ ions which have been used in the above recalled studies are unable to induce R•R.Y triplexes in d(GA.TC) n sequences (29) while Zn 2+ ions do (30). In many cases Zn 2+ ions have proved to be particularly efficient in antiparallel triple helix stabilization (31)(32)(33). Therefore, we have investigated whether Zn 2+ ions could also favor the formation of a parallel triplex.…”