In this work, we examined the DNA sequence preference of gamma-radiation-induced DNA damage in purified DNA sequences after heat treatment. DNA was fluorescently end-labeled and gamma-radiation-induced DNA cleavage was examined using capillary electrophoresis with laser-induced fluorescence detection. Our findings provide evidence that gamma-radiation-induced DNA damage to end-labeled DNA is nonrandom and has a sequence preference. The degree of cleavage was quantified at each nucleotide, and we observed that preferential cleavage occurred at C nucleotides with lesser cleavage at G nucleotides, while being very low at T nucleotides. The differences in percentage cleavage at individual nucleotides ranged up to sixfold. The DNA sequences surrounding the most intense radiation-induced DNA cleavage sites were examined and a consensus sequence 5'-AGGC*C (where C* is the cleavage site) was found. The highest intensity gamma-radiation-induced DNA cleavage sites were found at the dinucleotides, 5'-GG*, 5'-GC*, 5'-C*C and 5'-G*G and at the trinucleotides, 5'-GG*C, 5'-TC*A, 5'-GG*G and 5'-GC*C. These findings have implications for our understanding of ionizing radiation-induced DNA damage.

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Cisplatin is widely used as a cancer chemotherapeutic agent and this review covers the mechanism of action of cisplatin, cellular resistance to cisplatin, the genomic location of cisplatin adducts and the properties of DNA-targeted Pt complexes. A particular focus of this review is the interaction of Pt compounds with DNA. The technology involved in determining Pt-drug/DNA interactions has advanced and permits clearer views of this process. In particular, molecular biological techniques permit a more accurate and precise determination of the sequence specific preference of Pt adduct formation. Prospects for the sequence specific genome-wide determination of Pt adduct formation using next-generation sequencing are also discussed. Cisplatin analogues that are targeted to DNA via an attached DNA-affinic moiety are potentially beneficial anti-tumour agents. In particular the 9-aminoacridine Pt complexes possess a number of important characteristics, including activity against cisplatin-resistant cells. Their ability to circumvent resistance due to increased DNA repair may allow these DNA-targeted analogues to avoid many of the drawbacks associated with current clinical oncology treatment. This ability is thought to be due to their altered DNA sequence specificity, compared with cisplatin, where Pt adduct formation for the 9- aminoacridine Pt complexes was shifted away from consecutive guanines towards 5'-CG and 5'-GA dinucleotide sequences. Evidence for this evasion of repair processes and avoidance of cellular cisplatin resistance was found for 9-aminoacridine Pt complexes in studies with cisplatin resistant cells. The prospects for clinical use of these DNA-targeted anti-tumour agents were evaluated.

The aim of this paper was to investigate the sequence preference of ionizing radiation (IR)induced DNA damage as assessed by a linear amplification/polymerase stop (LA/PS) assay. The LA/PS assay is able to detect a wide range of IR-induced DNA lesions and this technique was utilized to quantitatively determine the preferential sites of gamma irradiation-induced DNA lesions in three different DNA sequences. Materials and methods: This analysis was performed on an automated DNA sequencer with capillary electrophoresis and laser-induced fluorescence detection. Results: The main outcome of this study was that G nucleotides were preferentially found at IRinduced polymerase stop sites. The individual nucleotides at the IR-induced DNA damage sites were analyzed and a consensus sequence of 5 0-GG à (where à indicates the damaged nucleotide) was observed. In a separate method of analysis, the dinucleotides and trinucleotides at the IR-induced DNA damage sites were examined and 5 0-GG à and 5 0-G à G dinucleotides and 5 0-GG à G trinucleotides were found to be the most prevalent. The use of the LA/PS assay permits a large number of IRinduced DNA lesions to be detected in the one procedure including: double-and single-strand breaks, apurinic/apyrimidinic sites and base damage. Conclusions: It was concluded that 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G) and the degradation products of 8-oxoG were possibly the main lesions detected. To our knowledge, this is the first occasion that the DNA sequence preference of IR-induced DNA damage as detected by a LA/PS assay has been reported.

Ionising radiation (IR) is known to induce a wide variety of lesions in DNA. In this review, we compared three different techniques that examined the DNA sequence preference of IR-induced DNA damage at nucleotide resolution. These three techniques were: the linear amplification/polymerase stop assay, the end-labelling procedure, and Illumina next-generation genome-wide sequencing. The DNA sequence preference of IR-induced DNA damage was compared in purified DNA sequences including human genomic DNA. It was found that the DNA sequence preference of IR-induced DNA damage identified by the end-labelling procedure (that mainly detected single-strand breaks) and Illumina next-generation genome-wide sequencing (that mainly detected double-strand breaks) was at C nucleotides, while the linear amplification/polymerase stop assay (that mainly detected base damage) was at G nucleotides. A consensus sequence at the IR-induced DNA damage was found to be 5 -AGGC*C for the end-labelling technique, 5 -GGC*MH (where * is the cleavage site, M is A or C, H is any nucleotide except G) for the genome-wide technique, and 5 -GG* for the linear amplification/polymerase stop procedure. These three different approaches are important because they provide a deeper insight into the mechanism of action of IR-induced DNA damage.The hydroxyl radical can cause both SSBs and DSBs via reaction with the deoxyribose sugar of DNA [15,33,34]. IR also induces DNA damage to the bases in DNA. The purine bases are damaged to a greater extent than the pyrimidines [28,35,36]. The major purine lesions induced by IR are: 8-oxo-7,8-dihydroguanine (8-oxoG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G), 8-oxo-7,8-dihydro-2 -deoxyadenosine (8-oxoA) and 4,6-diamino-5-formamidopyrimidine (Fapy-A). The major pyrimidine lesions are the thymine glycols that are the cis-and trans-diasteromers of 5,6-dihydroxy-5,6-dihydrothymine (Thy-Gly) [37,38].Apurinic/apyrimidinic (also known as abasic) sites are also produced by IR and can be identified by a number of enzymes [39][40][41][42][43][44][45][46]. These apurinic/apyrimidinic-specific enzymes can be used to detect the level of modified bases after IR treatment [33,34,47].The IR-induced damage site can be several nucleotides from the initial lesion [48,49]. A transfer process can occur whereby an electron loss centre (hole) can move along the DNA until it reaches a site with the lowest ionisation energy which is usually the guanine base [50,51]. In DNA with GG and GGG sequences, the hole is located at the 5 -G in a run of consecutive guanine nucleotides [52][53][54][55][56][57][58][59].It has been estimated that over 100 oxidatively generated DNA lesions and modifications are induced by IR [12]. A number of these lesions can be formed along the radiation track and thus can contribute to the complexity of IR-induced DNA damage. Techniques to Determine the Sequence Preference of IR-Induced DNA DamageThere are three techniques that have recently examined the DNA sequence preference of IR-induced DNA damage at nucleo...