Photoinduced Single Strand Breaks and Intrastrand Cross-Links in an Oligonucleotide Labeled with 5-Bromouracil

Abstract

5-Bromouracil (BrU) is photoreactive toward near UVB photons and can be introduced into genomic DNA during its biosynthesis in cells. However, PCR seems to be a simpler approach, which can be used to obtain labeled DNA similar to that synthesized within the cell. In the current work, PCR has been employed and optimized in order to substitute all thymines (besides those present in starters) with BrU in the dsDNA fragment of 80 base pairs (bp) in length. The modified oligonucleotide was irradiated with 300 nm photons in a buffered aqueous solution (pH = 7) and digested with a cocktail of enzymes specific to the phosphodiester bond cleavage. Initially, the extent of damage in the intact photolyte was measured with DHPLC. Then, the digested reaction mixture was subjected to HPLC and MS analyses and, in addition to the formation of 5-bromo-2'-deoxuyridine, which proves the occurrence of single strand breaks (SSBs) due to irradiation, U∧U and U∧C dimers were found, whose molecular structure was confirmed by MS/MS analysis. Although the abundance of such tandem lesions is lower than that of the SSB type, they pose a potent threat to genome integrity. Thus, our findings shed new light on the photosensitizing properties of BrU toward DNA.

5-Bromo-2′-deoxycytidine—a potential DNA photosensitizer

“…Hence, we employed the PCR reaction in order to obtain a labeled DNA fragment in which all the 2′-deoxycytidines, besides those present in the primers, are substituted with BrdC. A similar approach to the labeled DNA synthesis was used in the past by us, 16,21 as well as by other groups. 22 In comparison to our recent studies on the BrdU-labeled dsDNA fragment, 16 and also in the current studies, we amplified the same part of the pUC19 plasmid by PCR (for the PCR protocol, see ESI †).…”

“…Hence, changes in the absorbance due to a change in the system's constitution with the time of irradiation should not significantly perturb the quantum yield calculated for the first minute of exposure. It is worthy of emphasis that the quantum yield calculated for the same 80-mer sequence labeled with BrdU was as much as 8.4 × 10 −3 (measured for 1 min irradiation, i.e., the exposure time for which the degree of degradation versus irradiation time displays almost perfect linearity), 16 which is ca. 3-fold larger than that for DNA labeled with BrdC.…”

“…By the same analogy, one can assume that substitution of Tris-HCl by a buffer lacking hydrogens like phosphate or cacodylate buffer would have no effect on the studied process as we demonstrated some time ago. 16 Looking at Fig. 1, one can identify seven "hot spots", i.e., sequences in which BrdC is separated from the guanine located in the same or complementary strand by 1-2 AT base pairs.…”

“…In the current work, we study the UVB damage to a doublestranded oligonucleotide, multiply substituted with 5-bromo-2′-deoxycytidine, in order to assess its photosensitizing properties. By comparison to the BrdU photosensitizer whose main mode of action is related to long range photoinduced ET, 16 we assumed that BrdC should possess similar properties. Employing the PCR method, we synthesized an 80 bp DNA fragment in which all but the dC nucleosides present in the primers were replaced by BrdC.…”

Reactivity Pattern of Bromonucleosides Induced by 2-Hydroxypropyl Radicals: Photochemical, Radiation Chemical, and Computational Studies

“…In a double-stranded DNA, such a radical abstracts a hydrogen atom from the sugar residue or interacts with the adjacent bases, leading to serious DNA damage as strand breaks and intra-or interstrand crosslinks. 49 The Journal of Physical Chemistry B Article…”