Detection ofDNA damage: effect of thymidine glycol residues on the thermodynamic, substrate and interfacial acoustic properties of oligonucleotide duplexes

Abstract

Thymidine glycol residues in DNA are biologically active oxidative molecular damage sites caused by ionizing radiation and other factors. One or two thymidine glycol residues were incorporated in 19- to 31-mer DNA fragments during automatic oligonucleotide synthesis. These oligonucleotide models were used to estimate the effect of oxidized thymidines on the thermodynamic, substrate and interfacial acoustic properties of DNA. UV-monitoring melting data revealed that modified residues in place of thymidines destabilize the DNA double helix by 8-22 degrees C, depending on the number of lesions, the length of oligonucleotide duplexes and their GC-content. The diminished hybridizing capacity of modified oligonucleotides is presumably due to the loss of aromaticity and elevated hydrophilicity of thymine glycol in comparison to the thymine base. According to circular dichroism (CD) data, the modified DNA duplexes retain B-form geometry, and the thymidine glycol residue introduces only local perturbations limited to the lesion site. The rate of DNA hydrolysis by restriction endonucleases R.MvaI, R.Bst2UI, R.MspR9I and R.Bme1390I is significantly decreased as the thymidine glycol is located in the central position of the double-stranded recognition sequences 5'-CC / WGG-3' (W = A, T) or 5'-CC / NGG-3' (N = A, T, G, C) adjacent to the cleavage site. On the other hand, the catalytic properties of enzymes R.Psp6I and R.BstSCI recognizing the similar sequence are not changed dramatically, since their cleavage site is separated from the point of modification by several base-pairs. Data obtained by gel-electrophoretic analysis of radioactive DNA substrates were confirmed by direct spectrophotometric assay developed by the authors. The effect of thymidine glycol was also observed on DNA hybridization at the surface of a thickness-shear mode acoustic wave device. A 1.9-fold decrease in the rate of duplex formation was noted for oligonucleotides carrying one or two thymidine glycol residues in relation to the unmodified analog.

Thymidine Glycol: The Effect on DNA Structure and DNA Binding by Site-Specific Proteins

“…The CD spectra of the samples dissolved to ~2 µM concentration in buffer С were measured in a quartz cuvette The hyperchromic effect accompanying the double helix melting; relatively low h 260 value is explained by the presence of single-stranded prominent (4-and 2-nucleotides) termini flanking the duplex core, which absorb the UV light at 260 nm, but do not contribute to secondary structure unfolding. These data are already published in [15].…”

“…In the control experiments, R.SsoII was added to DNA duplexes not treated with M.SsoII. We have shown earlier that R.SsoII demonstrated an increased catalytic activity towards a Tg-containing duplex compared to a non-modified control duplex [15]. This feature provides high sensitivity for the analysis performed using R.SsoII.…”

“…The analysis proved that a single Tg lesion did not result in oligonucleotide decomposition at elevated temperatures (data not shown). Thus, the increase in optical density at 260 nm of the DNA duplexes was associated only with the double helix melting as mentioned in [15].…”

“…The oligonucleotides containing a single Tg lesion were synthesized by the standard phosphoramidite chemistry using the 5R,6S Tg phosphoramidite building block [15]. Since Tg is labile under alkaline conditions, we applied a short cycle of ammonia treatment at room temperature for oligonucleotide deprotection (see Materials and Methods).…”

“…Up to date, many researches are dedicated to the Tg influence on the activity of various DNA polymerases and to the Tg removal from DNA by repair enzymes (reviewed in [14]), but there is a lack of information about Tg impact onto proteins which recognize specific DNA sites. The only research addressing this question is our paper [15] where effect of Tg on the rate of DNA hydrolysis by several restriction endonucleases (MvaI, Bst2UI, MspR9I, Bme1390I, Psp6I, BstSCI, EcoRII, and SsoII) has been studied.…”