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Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 2005

Translin Unfolds DNA Quadruplex Formed by Telomeric d(TTAGGG) Repeats

Translin is a single-stranded DNA and RNA binding protein that has been highly conserved in evolution. Previous data suggested that the human and the mouse Translins, which are virtually identical, could be involved in several biological processes, including chromosomal translocations, control of mRNA translation and transport, and RNA interference. However, a definition of the functions of Translin in precise molecular terms is lacking. Recently, some of us demonstrated that the human Translin selectively binds single-stranded G-rich DNA sequences. In particular, it was found that Translin binds with a high affinity single-stranded microsatellite repeats, d(GT)n, human telomeric repeats, d(TTAGGG)n and Tetrahymena telomeric repeats, d(GGGGTT)n. These data led to the suggestion that Translin might be involved in recombination at d(GT)n·d(AC)n microsatellites and in telomere metabolism (1, 2). In other experiments, it was shown that relatively low concentrations of Translin stimulate DNA primer extension by telomerase (2). One possibility raised by this finding was that Translin might stimulate binding of telomerase to single-stranded telomeric overhangs by unwinding secondary and tertiary structures formed by the telomeric repeats.

Here we describe experiments aimed at addressing this possibility. Circular dichroism (CD) at a wavelength range of 260-320 nm was used for monitoring conformational changes that occur in the oligonucleotide d(TTAGGG)5 in the presence of 0.10 M NaCl upon the addition of Translin at increasing concentrations. We observed a characteristic positive band around 295 nm in the spectra of the oligonucleotides d(TTAGGG)4 and d(TTAGGG)5. The appearance of this band indicated that intramolecular antiparallel quadruplexes were formed in both cases. Hence, a d(TTAGGG) sequence remained single-stranded in the latter oligonucleotide. The figure shows changes that occurred in the CD signal of the oligonucleotide d(TTAGGG)5 at the wavelengths 300 nm and 265 nm, as a function of Translin concentration. Evidently, the signal decreased in both wavelengths up to the 1:1 stoichiometry point. Comparison to the CD spectra of the oligonucleotides d(TTAGGG)4 and d(TTAGGG)2 (taken in the absence of Translin) supported the presence in the Translin-d(TTAGGG)5 complexes of a hairpin formed by association of two d(TTAGGG) repeats, rather than a G4 quadruplex. Based on these data, we concluded that the oligonucleotide d(TTAGGG)5 in the complexes comprised a single hairpin and three d(TTAGGG) repeats. Thus, it appears that upon binding the oligonucleotide d(TTAGGG)5, Translin disrupts one of the two preexisting hairpins and creates a structure consisting of a single hairpin and three single-stranded d(TTAGGG) repeats. It should also be noted that neither stoichiometric Translin binding, nor unfolding were observed in similar assays of d(TTAGGG)4 quadruplex or d(TTAGGG)2 hairpin. Hence, the single-stranded d(TTAGGG) overhang in the d(TTAGGG)5 structure is required for the unfolding of one hairpin. We suggest that such unfolding could cause the stimulation in telomerase activity mentioned above.


Acknowledgement

The study was supported by the RFBR grant N 04-04-49618.

References and Footnotes
  1. E. Jacob, L. Pucshansky, E. Zeruya, N. Baran, and H. Manor. J. Mol. Biol. 344,939-950 (2004).
  2. S. Cohen., E. Jacob, and Manor, H. Biochim. Biophys. Acta. 1679, 129-140 (2004).

Dmitry Kaluzhny1
Orly Laufman2
Edward Timofeev1
Olga Borisova1
Haim Manor2
Anna Shchyolkina1,*

1Engelhardt Institute of Molecular Biology RASc
Vavilova 119991 Moscow, Russia
2Department of Biology
Technion-Israel Institute of Technology
Haifa 32,000, Israel

*Phone: 7095-1359789
Fax: 7095-1351405
Email: ashchyolkina@mail.ru