Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Effect of Guanine Substitutions in Human Telomeric G3 (T2 AG3)3 DNA Sequence
In addition to the well-known Watson-Crick double helix, DNA can form other structures. One of them is a four-stranded quadruplex, formation of which was also acknowledged in in vivo conditions. It was suggested that the presence of quadruplexes in e.g. telomeric region has a significant biological importance.
We have studied structural properties of the human telomeric quadruplex formed by G3 (T2 AG3)3 and related sequences, in which each guanine base was one-by-one replaced by adenine. In the next step, we have studied sequences, in which two, or even four guanines were replaced by adenine. These sequences were studied in the presence of sodium or potassium ions. Using CD spectroscopy, UV thermal stability measurements and polyacrylamide gel electrophoresis we found that none of the substitutions hindered the formation of the antiparallel quadruplex formed by the unsubstituted sequence in sodium solutions. However, the effect of substitution differed depending on the position of the guanine replaced. The middle quartet of the antiparallel basket scaffold was the most sensitive and led to the least stable structures. With other sequences, the effect of substitution depended on the position and also on the syn/anti glycosidic bond orientation of the appropriate guanosine in the original quadruplex structure. In the case of the multiple A for G substitutions, the G3 (T2 AG3)3 quadruplex was most destabilized by the G:G:A:A tetrad, in which the adenosines substituted syn guanosines. Interestingly, unlike with G3 (T2 AG3)3, no structural transitions were observed with the A-containing analogs of the sequence when sodium ions were replaced by potassium ions. The basic quadruplex topology remained antiparallel for all modified sequences in both salts. As in vivo misincorporation of A for a G in the telomeric sequence is possible and potassium is a physiological salt, these findings may be biologically important.
I our next studies we have compared the effect of the G to A substitutions in the human telomere sequence with 8-oxoguanine substituted samples or samples containing guanine apurinic sites. Data obtained from our study show a noticeable trend: it is not the type of the lesion but the position of the modification determines the effect on the conformation and stability of the quadruplex.
This research has been supported by Grant OP VK (CZ.1.07/2.3.00/30.0019), Grant P205/12/0466 from the Grant Agency of the Czech Republic and by the project ‘CEITEC – Central European Institute of Technology’ (CZ.1.05 ⁄ 1.1.00 ⁄ 02.0068) from the European Regional Development Fund.
Sagi, J., Renciuk, D., Tomasko, M., Vorlickova, M. Quadruplexes of human telomere DNA analogs designed to contain G:A:G:A, G:G:A:A and A:A:A:A tetrads. Biopolymers 93, 880-886
Skolakova, P., Bednarova, K., Vorlickova, M., Sagi, J. Quadruplexes of human telomere dG3(TTAG3)3 sequences containing guanine abasic sites. Biochem Biophys Res Commun 399, 203–208.
Vorlickova, M., Tomasko, M., Sagi, A.J., Bednarova, K., Sagi, J. 8-oxoguanine in a quadruplex of the human telomere DNA sequence. FEBS J. 279, 29-39
Martin Tomaško 1
1 Institute of Biophysics