Background: The concept of chromatin domains attached to the nuclear matrix is being revisited, with nucleus described as a set of topologically associating domains. The significance of the tightly bound to DNA proteins (TBP), a protein group that remains attached to DNA after its deproteinization should be also revisited, as the existence of these interactions is in good agreement with the concept of the topologically associating domain. The work aimed to characterize the DNA component of TBP isolated from barley seedlings.
Methods: The tight DNA-protein complexes from the first leaves, coleoptiles, and roots of barley seedlings were isolated by purification with chromatography on nitrocellulose or exhaustive digestion of DNA with DNase I. Cloning and transformation were performed using pMOSBBlue Blunt Ended Cloning Kit. Inserts were amplified by PCR, and sequencing was performed on the MegaBace 1000 Sequencing System. The BLAST search was performed using sequence databases at NCBI, CR-EST, and TREP and Ensembl Plants databases. Comparison to MAR/SAR sequences was performed using http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi database. The prediction of G quadruplexes (GQ) was performed with the aid of R-studio library pqsfinder. CD spectra were recorded on a Chirascan CS/3D spectrometer.
Results: Although the barley genome is AT-rich (43% of GC pairs), most DNA fragments associated with TBP were GC-rich (up to 70% in some fractions). Both fractionation procedures yielded a high proportion of CT-motif sequences presented predominantly by the 16-bp CC(TCTCCC)2 TC fragment present in clones derived from the TBP-bound DNA and absent in free DNA. BLAST analysis revealed alignment with different barley repeats. Some clones, however, aligned with both nuclear and chloroplast structural genes. Alignments with MAR/SAR motifs were very few. The analysis produced by the pqsfinder program revealed numerous potential quadruplex-forming sites in the TBP-bound sequences. A set of oligonucleotides containing sites of possible GQs were designed and ordered. Three of them represented the minus strand of the CT-repeat. Two were derived from sequences of two clones of nitrocellulose retained fraction from leaves and contained GC-rich motifs different from the CT motif. Circular dichroism spectroscopy revealed profound changes in spectra when oligonucleotides were incubated with 100 mM KCl. There was either an increase of positive band in the area of 260 nm or the formation of a positive band at 290 nm. In the former case, changes are typical for parallel G-quadruplexes and, in the latter, 3 + 1 structures.
Discussion: The G-quadruplexes anchor proteins are probably involved in the maintenance of the topologically associated domain structure.
Keywords: Barley; Deproteinisation-resistant DNA-protein complexes; G-quadruplexes; GC-rich DNA.
©2020 Sjakste et al.