Book of Abstracts: Albany 2003
June 17-21 2003
Formation of the Parallel Recombination-type Triplex by Human Telomeric Sequences
Three-stranded nucleic acid alignments occur in various biological processes, such as recombination, transcription and formation of telomeres, specifically the mammalian t-loops (1). In all these cases, the two identical strands are parallel to each other, while the complementary strand is antiparallel to the first two. Such an orientation of the DNA and RNA strands is consistent, in principle, with formation of a parallel (recombination-type) ?R-triplex? that can accommodate any nucleotide sequence (2).
In the human telomere, a single TTAGGG hexanucleotide in the ss overhang next to the ds telomeric DNA is sufficient for stabilization of the TRF2-induced t-loop (3). In contrast, an artificial CCCTAA overhang (from the complementary strand) forms a t-loop much less effectively. What are the structural and/or thermodynamic features, responsible for such a difference between the two sequences? To address this question, we studied intramolecular triplexes containing these hexamers, which potentially can serve as the transient intermediates for the TRF2-stabilized t-loops. Earlier we developed a method for monitoring formation and dissociation of the R-triplex using the fluorescent base analog 2-aminopurine (2AP) incorporated in the third non-WC strand (4). Here, we show that (i) both the GGG-overhang of the human telomere and the artificial CCC-overhang can form an R-triplex, and (ii) the thermodynamics of formation of the R-triplex formed by the human telomeric sequence drastically depends on the solution conditions.
Oligonucleotides 5'-GGTT-2AP-GGG TTTT CCCTAACCG GAA CGGTTAGGG-3' (Tel-G) and 5'-CCCTAACCG GAA CGGTTAGGG TTTT CCCT-2AP-ACC-3' (Tel-C), where 2AP is for 2-aminopurine, were studied in 10 mM Tris HCl buffer, pH 7.6, and 0.5 M LiCl or 2 mM MnCl2. The oligonucleotides were designed such that a GAA linker connects the antiparallel Watson-Crick strands, while a flexible TTTT loop links them to the third strand to the duplex; the identical hexanucleotide telomeric repeats are underlined. Fluorescence-detected formation of the single 2AP*(T·A) triplet was evidenced earlier to reflect formation of the R-triplex. By measuring the 2AP fluorescence we showed that both Tel-G and Tel-C are able to ?fold back? and form the protein-free R-triplex. Additionally, the temperature-dependent association of the third strand with the duplex part was monitored by fluorescence anisotropy of an ethidium bromide probe. Thermodynamics of the R-triplex formation was inferred using the temperature dependence of the 2AP fluorescence.
In the presence of Mn2+ the thermodynamic parameters of the two triplexes, Tel-G and Tel-C, were found to be similar, the former being more stable by 1.5 kJ·mol-1 at 0°C. Unlike Tel-C, however, stability of the triplex Tel-G is markedly influenced by the counterion type. In the Li+ buffer, the dissociation enthalpy of the Tel-G triplex (69± 12 kJ·mol-1) is substantially larger than that of Tel-C (39± 2 kJ·mol-1). The dissociation entropy of the Tel-G triplex is also larger than that of the Tel-C triplex, thereby decreasing melting temperature of the former (from 22°C in the Mn2+ buffer to 8°C in Li+ buffer). We interpret these differences based on the sequence-dependent structure of the R-triplex (3), suggesting that penetration of the hydrated Li+ ions into one of the two major ?subgrooves? could stabilize the more regular Tel-G triplex stronger than the less regular Tel-C triplex. The latter assumption follows from the different geometries of the G*G:C and the C*C:G triplets (2).
The sequence-dependent thermodynamic properties of the triplex structure would affect a dynamic equilibrium between R-triplex (2) and displacement D-loop. (The D-loop is assumed to form in telomeres (1) after displacement of the identical duplex strand by the invading overhang.) In turn, the above equilibrium is critical for the ss-ds DNA recognition process. Overall, the ?natural? Tel-G triplex demonstrated a much greater sensitivity to the environmental conditions compared to the ?artificial? triplex Tel-C. We suggest this lability of the ?natural? triplex might be utilized by the TRF2 protein for a kinetic stabilization of the complex with three strands of the telomeric DNA.
The study was supported with NATO SA (LST.CLG.978296) and RFBR 01-04-48561 grants.
Anna K. Shchyolkina1,*
1Engelhardt Institute of Molecular Biology RASc