Investigating the Biochemical Impact of DNA Damage with Structure-Based Probes: Abasic Sites, Photodimers, Alkylation Adducts, and Oxidative Lesions

Abstract

DNA sustains a wide variety of damage, such as the formation of abasic sites, pyrimidine dimers, alkylation adducts, or oxidative lesions, upon exposure to UV radiation, alkylating agents, or oxidative conditions. Since such damage may be acutely toxic or mutagenic and potentially carcinogenic, it is of interest to gain insight into how their structures impact biochemical processing of DNA, such as synthesis, transcription, and repair. Lesion-specific molecular probes have been used to study polymerase-mediated translesion DNA synthesis of abasic sites and TT dimers, while other probes have been developed to specifically investigate the alkylation adduct O 6 -Bn-G and the oxidative lesion 8-oxo-G. In this review recent examples of lesion-specific molecular probes are surveyed; their specificities of incorporation opposite target lesions compared to unmodified nucleotides are discussed, and limitations of their applications under physiologically relevant conditions are assessed.Within years of the discovery that DNA 1 was responsible for transmitting hereditary information (1), and before publication of the double helical structure of DNA (2), researchers began to probe its susceptibility to various types of damage (3). Exposure of DNA to UV radiation, alkylating agents, and reactive oxidative species leads to the formation of abasic sites, pyrimidine dimers, alkylation adducts, and oxidative damage products. Each of these types of lesions can be mutagenic, sometimes carcinogenic, and this chemical process, therefore, is highly relevant to cellular biology and disease. For example, UV radiation damageinduced skin cancer was estimated to result in over one million new cases of the disease in the United States in 2008 (4). Further, DNA alkylation by tobacco-derived carcinogens contributes to lung cancer, which is estimated will result in about 160,000 deaths in the United States in 2009 (5,6). These examples of the biological consequences of DNA damage underscore the continued need for understanding the underlying molecular mechanisms.

Fluorescence Properties of 5‐(5,6‐Dimethoxybenzothiazol‐2‐yl)‐2′‐deoxyuridine (dbtU) and Oligodeoxyribonucleotides Containing dbtU

“…[10] Some selective detection methods for DNA damage by chemical reagents have recently been reported. [11] However, trace amounts of excess fluorescent and/or colorimetric probe remain even after extensive washing, which interfere with the analysis by giving higher background signals. We recently reported a new concept for simple fo Ura detection in DNA using a fluorogenic reagent, 2-amino-4,5-dimethoxythiophenol, which has no fluorescence before reaction with the target fo Ura in DNA.…”

A chemical toxicity sensor based on the electrochemiluminescence quantification of apurinic/apyrimidinic sites in double-stranded DNA monolayer

“…ELISA assay is simple to operate, but the antibodies used in the assay to recognize AP sites may lack specificity. More recently, research work has focused on bioassays and biosensors employing structure-based chemical probes for AP site recognition and detection, which are either non-covalent or covalent reaction type probes [7,8]. After the formation of an AP site in DNA, a cavity is created that can readily accommodate a small molecule with complementary shape and binding forces.…”

Oligonucleotide probes containing pyrimidine analogs reveal diminished hydrogen bonding capacity of the DNA adduct O6-methyl-G in DNA duplexes

“…Central to their function is their inclusion of nucleoside analogs that preferentially pair opposite or react with the targeted DNA lesion. [4][5][6][7][8][9] Two types of DNA adduct-directed hybridization probes have been reported for O 6 -alkyl-G: a chemically reactive probe and hydrophobic intercalating probes. 4,7,10 For the chemically reactive hybridization probe system for detecting O 6 -Me-G, 8 an oligonucleotide containing 6-thio-G modified with an alkynylated enone was annealed to a complementary target strand; the enone moiety was then transferred to the N 2 -position of the complementary O 6 -Me-G, but not to that of a complementary G. Selectivity in this case was attributed to the enhanced nucleophilicity of O 6 -Me-G compared to G. The appended alkyne was also labeled with a fluorescent azide via Click chemistry for spectroscopic detection.…”