Book of Abstracts: Albany 2005
Recombination R-triplex: H-bonds Contribution to Stability as Revealed with Minor Base Substitutions for Adenine
A variety of cellular processes involves alignment of three nucleic acids strands, in which the third strand (DNA or RNA) is identical and parallel oriented to one of the DNA duplex strands. Earlier, using fluorescence structural probes incorporated in the third strand (1) we have shown that a DNA sequence comprising four natural bases cooperatively forms a self-folded recombination-like structure with the third strand tightly attached to the duplex, consistent with the R-triplex structure (2, 3). Here, we further extended this approach, placing the reporting 2-aminopurine strand-specifically either in the duplex or in the third strand.
(i) In this way, we proved formation of the equilibrium triplex rather than a ?branch migration? structure with dynamic exchange of the two homologous strands.
(ii) Substituting minor bases for adenine (e.g., 2,6-diaminopurine and 7-deazaadenine) we tested and confirmed the H-bonding scheme of the A*(T·A) R-type triplet suggested earlier (2). In particular, the adenine substitutions expected to provide an additional H-bond in the A*(T·A) triplet, lead to an increasingly stable triplex structure. By contrast, substitutions consistent with the decrease in number of H-bonds (2) destabilized the triplex.
(iii) Remarkably, the changes in the triplex formation enthalpy and free energy proved to be linearly dependent on the change in number of H-bonds according to the postulated A*(T·A) triplet scheme (2). The average enthalpy cost of a single H-bond between the third strand and the duplex was found to be 18 kJ mol-1, a surprisingly high value.
(iv) At the same time, the whole formation enthalpy of the 10 nt-long intramolecular triplex was estimated to be -100 kJ mol-1. That is, the recombination-like R-triplex is relatively unstable, which makes it an ideal candidate for a transient intermediate in homologous recombination, t-loop formation at the mammalian telomere ends, and short RNA invasion into the duplex.
Finally, we hypothesize that the observed significant energy advantage of a ?correct? base in the third strand opposite the Watson-Crick base pair, may be a powerful mechanism securing selectivity of recognition of nucleic acid duplex with the single strand and proper alignment of the third strand along the duplex.
References and Footnotes
1Engelhardt Institute of Molecular Biology