Conformational diversity of single‐stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases

Abstract

Repetitive extragenic palindrome (REP)—associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5′-G and 3′-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 585–596, 2015.

Structural variability of CG-rich DNA 18-mers accommodating double T–T mismatches

“…2 and Supplementary Figs. S1-S3) confirmed the results of our previous study (Charnavets et al, 2015) showing that CG-rich, near-palindromic REPs can adopt structures other than hairpins. The results indicate dynamic equilibria between the right-handed form(s) and tetraplex architectures formed by two or four strands; the possible topologies are outlined in Fig.…”

“…Both Hpar-18 and Chom-18 can acquire several molecular architectures, as outlined in Fig. 1, and our solution data confirm the previous observation (Charnavets et al, 2015) that oligonucleotides with REP-related sequences adopt multiple conformations in dynamic temperature-and solution-dependent equilibria. In the crystal phase, these DNA 18-mers form double helices with two successive T-T mismatches in the center of the duplexes.…”

“…S1a). However, as discussed in greater detail in our previous work (Charnavets et al, 2015), such spectral features are not fully compatible with the CD spectra of pure 'classic' intramolecular antiparallel tetraplexes. The CD spectrum of a folded unimolecular or bimolecular antiparallel quadruplex would display a positive peak near 295 nm, which is often accompanied by a strong negative peak near 265 nm.…”

“…Hairpin conformations are considered to be biologically relevant for the recognition of REP by RAYT proteins, as revealed by the only known structure of a REP-RAYT complex (Messing et al, 2012). A previous biophysical study in solution (Charnavets et al, 2015) showed that REPs from various bacterial species can also adopt conformations other than hairpins. Such structural and conformational variability of the REP sequences would be essential in the genomic context in order to participate in interactions with RAYT variants.…”

The Cyanobacterial Ribosomal-Associated Protein LrtA from Synechocystis sp. PCC 6803 Is an Oligomeric Protein in Solution with Chameleonic Sequence Properties

“…However, it is important to note that the presence of deoxyribonucleotides did not affect the spectroscopic signals either of both fluorescence and far-UV CD, because DNA is spectroscopically silent in fluorescence, and in the far-UV region of CD spectra between 210–240 nm, deoxyribonucleotides do not absorb [ 44 , 45 ]. It is also interesting to note that deoxyoligonucleotides in aqueous solution or intact DNA show a small or slightly positive ellipticity around 222 nm [ 45 , 46 , 47 , 48 , 49 ] where we have carried out the study of the CD biophysical properties of the protein (see Results section).…”