DHPLC and MS studies of a photoinduced intrastrand cross-link in DNA labeled with 5-bromo-2′-deoxyuridine

Wiczk, Justyna; Miloch, Justyna; Rak, Janusz

doi:10.1016/j.jphotobiol.2013.11.004

Cited by 4 publications

(8 citation statements)

References 41 publications

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“…BrdU‐labeled DNA is sensitive to both ionizing and UV radiation 17–20. 23–28 Although the mechanisms of SSB formation triggered by SEs and UV photons are quite different,17, 24, 25, 27, 28 both damaging agents lead to SBs in 5‐BrdU‐labeled DNA. As using a UV source is far easier than using ionizing radiation, we decided to employ the former in order to validate our methodology.…”

Section: Methodsmentioning

confidence: 99%

Artificial Plasmid Labeled with 5‐Bromo‐2′‐deoxyuridine: A Universal Molecular System for Strand Break Detection

Żylicz-Stachula¹,

Polska²,

Skowron³

et al. 2014

ChemBioChem

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DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid.

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Section: Methodsmentioning

confidence: 99%

Artificial Plasmid Labeled with 5‐Bromo‐2′‐deoxyuridine: A Universal Molecular System for Strand Break Detection

Żylicz-Stachula¹,

Polska²,

Skowron³

et al. 2014

ChemBioChem

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“…In both cases, the debromination of the starting dinucleotide is involved in the concomitant addition of a hydroxyl group followed by its further photochemical and thermal degradation to a 5,5′-diuridynyl product. 82 79 The results obtained for longer sequences revealed that the photoirradiation of 40 bp length duplex DNA containing the 5′-GAAAA Br UA-3′ sequence resulted in the formation of the d(A[2−5]U) dimer, in which the C5 carbon atom of dU and the C2 carbon atom of dA were covalently bonded, 83 while UV irradiation of dsDNA (80 bp length) containing all thymines substituted with 5-BrdU led to the formation of two intrastrand cross-linked products: d(U[5−5]C) and d(U[5−5]U). These dimers were produced by the electronic excitation of BrdU followed by photocycloaddition.…”

Section: Uv-induced Damage To Dna Labeled Withmentioning

confidence: 99%

Mechanisms of Damage to DNA Labeled with Electrophilic Nucleobases Induced by Ionizing or UV Radiation

et al. 2015

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Hypoxia--a hallmark of solid tumors--makes hypoxic cells radioresistant. On the other hand, DNA, the main target of anticancer therapy, is not sensitive to the near UV photons and hydrated electrons, one of the major products of water radiolysis under hypoxic conditions. A possible way to overcome these obstacles to the efficient radio- and photodynamic therapy of cancer is to sensitize the cellular DNA to electrons and/or ultraviolet radiation. While incorporated into genomic DNA, modified nucleosides, 5-bromo-2'-deoxyuridine in particular, sensitize cells to both near-ultraviolet photons and γ rays. It is believed that, in both sensitization modes, the reactive nucleobase radical is formed as a primary product which swiftly stabilizes, leading to serious DNA damage, like strand breaks or cross-links. However, despite the apparent similarity, such radio- and photosensitization of DNA seems to be ruled by fundamentally different mechanisms. In this review, we demonstrate that the most important factors deciding on radiodamage to the labeled DNA are (i) the electron affinity (EA) of modified nucleoside (mNZ), (ii) the local surroundings of the label that significantly influences the EA of mNZ, and (iii) the strength of the chemical bond holding together the substituent and a nucleobase. On the other hand, we show that the UV damage to sensitized DNA is governed by long-range photoinduced electron transfer, the efficiency of which is controlled by local DNA sequences. A critical review of the literature mechanisms concerning both types of damage to the labeled biopolymer is presented. Ultimately, the perspectives of studies on DNA sensitization in the context of cancer therapy are discussed.

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“…This value is between the QYs measured for singly labeled oligonucleotides (QY=0.07), 21 where long-range electron transfer triggers the damage, and those characteristic of intrastrand dimer formation (QY=0.004). 24 Indeed, as demonstrated in the previous section, irradiation of the studied DNA fragment leads to two types of damage: 2′deoxyuridine coupled to a strand break, and intrastrand dimers. The former is formed as a result of longrange electron transfer (ET) between the photoexcited 5-bromuracil and a distant 5′-side guanine that ultimately converts BrdU into dU.…”

Section: Enzymatic Digestion Coupled To Lc-ms Analysismentioning

confidence: 70%

“…Very recently, we demonstrated that DHPLC is quite convenient for studies of DNA damage and appears to be more accurate and more sensitive than gel electrophoresis. 24 As a matter of fact, nondenaturing polyacrylamide gel electrophoresis employed against the irradiated samples shows no effect of irradiation (see Figure S1). This is in complete contrast to the DHPLC chromatograms depicted in Figure 2.…”

Section: Resultsmentioning

confidence: 95%

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

et al. 2014

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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.

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DHPLC and MS studies of a photoinduced intrastrand cross-link in DNA labeled with 5-bromo-2′-deoxyuridine

Cited by 4 publications

References 41 publications

Artificial Plasmid Labeled with 5‐Bromo‐2′‐deoxyuridine: A Universal Molecular System for Strand Break Detection

Artificial Plasmid Labeled with 5‐Bromo‐2′‐deoxyuridine: A Universal Molecular System for Strand Break Detection

Mechanisms of Damage to DNA Labeled with Electrophilic Nucleobases Induced by Ionizing or UV Radiation

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

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