Repetitive extragenic palindromic (REP) elements are short (around 20-30 bp long) DNA non-coding sequences. In genomes of various bacterial species, they occur in a high number of copies and are often clustered into bacterial interspersed mosaic elements (BIME). These sites serve as a binding platform for a diverse group of proteins. REPs are usually characterized by having conserved GTAG sequence on its 5’-end followed by GC-rich palindrome [1]. Recently a new class of transposases (REP associated tyrosine transposase – RAYT) was discovered. It is unusual in its ability to act strictly upon single-stranded DNA. RAYT gene is usually flanked by REP [2].
We analyzed selected sequences using circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and X-ray crystallography. CD spectra of all analyzed oligonucleotides show signal characteristic for guanosine-quadruplex with complex topology strongly indicating its presence in solution even in higher temperatures (around 80 °C).
In order to measure the value of melting temperature, we measured absorbance at 260 nm at a temperature range between 10 and 90 °C. To determine the number of contributing species we subjected the measured spectra to single-value decomposition. We observed that there are more than one species present in solution.
Three sequences have been crystallized – Hpar1-18mer, Chom-18mer and Chom-18mer bromouracil derivative. They crystallized within 2-4 days in hanging drop setup. Phase problem was solved using anomalous diffraction on bromine in the bromouracil derivative crystal, bromouracil was placed instead of thymine in position 9 in Chom-18mer. Oligonucleotides of both sequences form a duplex structure with TT-mismatch in the center. Both structures were determined at the crystallographic resolution of 2.7 Å.
LTAUSA18197 Design, development, and testing of bioinformatic tools for validation of experimental and computer molecular models in structural biology, biotechnology and pharmacy.
CIISB4HEALTH This publication was supported by the project Czech infrastructure for integrative structural biology for human health (CZ.02.1.01/0.0/0.0/16_013/0001776) from the ERDF.