STRUCTURE, STABILITY AND THERMODYNAMIC CHARACTERISTICS OF SINGLE STRANDED DNA IN REPETITIVE EXTRAGENIC PALINDROMIC ELEMENTS

T. Charnavets¹, J. Nunvar¹, I. Zuskova¹, J. Völker², B. Schneider¹

¹Institute of Biotechnology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic,
²Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Taylor Road Piscataway 610, NJ, USA

Repetitive extragenic palindromes (REPs) are small imperfect palindromic repeats of 20–40 DNA nucleotides in length which are found in many bacterial species at a high copy number [1]. They are important in the regulation of certain bacterial functions, such as Integration Host Factor recruitment and mRNA turnover. REPs consist of a GTAG tetranucleotide and a nearby self-complementary GC-rich sequence with the potential to form stable non canonical DNA secondary structures. Single strand hairpin stem loop conformations have been shown to provide preferable binding sites for the REP-associated tyrosine transposase (RAYT) from Escherichia coli [2]. The conserved tetranucleotide and palindrome of REP elements play a key role in REP recognition by RAYTs.

Genomic sequence analysis has identified numerous potential REPs elements in a number of different host bacterial species. REPs from Escherichia coli, Stenotrophomonas maltophilia and Cardiobacterium hominis were chosen to explore the possibility of non-canonical DNA structure formation in these sequences similar to that observed for REP of Escherichia coli. Spectroscopic and calorimetric melting studies of single stranded oligonucleotide REP sequences were performed to determine their conformation, stability and thermodynamic characteristics.

Stability of oligonucleotides conformations was ascertained from their temperature-induced melting transitions monitored by ultraviolet absorbance. Model dependent transition parameters were estimated from a van’t Hoff analysis of the melting curves and, in select cases, compared to model free calorimetric measurements. The conformational state of the oligonucelotides was assessed by circular dichroism spectroscopy (CD) and the concentration dependence of the melting temperature. Our data showed that oligonucleotides corresponding to REP recognition DNA sequences from different bacteria can adopt a range of non-canonical DNA conformations in solution, which may be important for RAYT recognition of REP elements.

1. Nunvar J, Huckova T, Licha I.: Identification and characterization of repetitive extragenic palindromes (REP)-associated tyrosine transposases: implications for REP evolution and dynamics in bacterial genomes. BMC Genomics 11:44, 10.1186/1471-2164-11-44 (2010).

2. Messing SA, Ton-Hoang B, Hickman AB, McCubbin AJ, Peaslee GF, Ghirlando R, Chandler M, Dyda F.: The processing of repetitive extragenic palindromes: the structure of a repetitive extragenic palindrome bound to its associated nuclease. Nucleic Acids Res. 40, 9964-9979 (2012).

Acknowledgements. This work is supported by grant CZ.1.07/2.3.00/30.0020 from the Ministry of Education of the Czech Republic (MSMT).