Crystal structure and biophysical characterization of selected REP sequences

Jakub Svoboda1, Petr Kolenko1,2, Tatsiana Charnavets1 and Bohdan Schneider1


1Laboratory of Biomolecular Recognition, Institute of Biotechnology of the Czech Academy of Sciences, v.v.i., Prumyslova 595, Vestec, 252 50, Czech Republic

2Dept. of Solid State Engineering, FNSPE Czech Technical University, Brehova 7, Prague 1, 11519, Czech Republic

 

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.

1.         Di Nocera, P.P., De Gregorio, E., Rocco, F. (2013) GTAG- and CGTC-tagged palindromic DNA repeats in prokaryotes. Bmc Genomics 14, 522

2.         Nunvar, J., Huckova, T., Licha, I. (2010) Identification and characterization of repetitive extragenic palindromes (REP)-associated tyrosine transposases: implications for REP evolution and dynamics in bacterial genomes. Bmc Genomics 11, 44.

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.