Comparison of Different Methods to Determine the DNA Sequence Preference of Ionising Radiation-Induced DNA Damage.

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 sequence preference of gamma radiation-induced DNA damage as determined by a polymerase stop assay.

Purpose: 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 IR-induced polymerase stop sites. The individual nucleotides at the IR-induced DNA damage sites were analyzed and a consensus sequence of 5'-GG* (where * 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'-GG* and 5'-G*G dinucleotides and 5'-GG*G trinucleotides were found to be the most prevalent. The use of the LA/PS assay permits a large number of IR-induced 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.

The genome-wide sequence preference of ionising radiation-induced cleavage in human DNA.

For ionising radiation (IR)-induced cellular toxicity, DNA cleavage is thought to be a crucial step. In this paper, the genome-wide DNA sequence preference of gamma radiation-induced cleavage was investigated in purified human DNA. We utilised Illumina short read technology and over 80 million double-strand breaks (DSBs) were analysed in this study. The frequency of occurrence of individual nucleotides at the 50,000 most frequently cleaved sites was calculated and C nucleotides were found to be most prevalent at the cleavage site, followed by G and T, with A being the least prevalent. 5'-C*C and 5'-CC* dinucleotides (where * is the cleavage site) were found to be the present at the highest frequency at the cleavage site; while it was 5'-CC*C for trinucleotides and 5'-GCC*C and 5'-CC*CC for tetranucleotides. The frequency of occurrence of individual nucleotides at the most frequently cleaved sites was determined and the nucleotides in the sequence 5'-GGC*MH (where M is A or C, H is any nucleotide except G) were found to occur most frequently for DNA that was treated with endonuclease IV (to remove blocking 3'-phosphoglycolate termini); and 5'-GSC*MH (where S is G or C) for non-endonuclease IV-treated DNA. It was concluded that GC-rich sequences were preferentially targeted for cleavage by gamma irradiation. This was the first occasion that an extensive examination of the genome-wide DNA sequence preference of IR-induced DSBs has been performed.

The Sequence Preference of Gamma-Radiation-Induced Damage in End-Labeled DNA after Heat Treatment.

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.

The sequence preference of DNA cleavage by T4 endonuclease VII.

The enzyme T4 endonuclease VII is a resolvase that acts on branched DNA intermediates during genetic recombination, by cleaving DNA with staggered cuts approximately 3-6 bp apart. In this paper, we investigated the sequence preference of this cleavage reaction utilising two different DNA sequences. For the first time, the DNA sequence preference of T4 endonuclease VII cleavage sites has been examined without the presence of a known DNA substrate to mask any inherent nucleotide preference. The use of the ABI3730 platform enables the cleavage site to be determined at nucleotide resolution. We found that T4 endonuclease VII cleaves DNA with a sequence preference. We calculated the frequency of nucleotides surrounding the cleavage sites and found that following nucleotides had the highest incidence: AWTAN*STC, where N* indicates the cleavage site between positions 0 and 1, N is any base, W is A or T, and S is G or C. An A at position -1 and T at position +2 were the most predominant nucleotides at the cleavage site. Using a Sequence Logo method, the sequence TATTAN*CT was derived at the cleavage site. Note that A and T nucleotides were highly preferred 5' to the cleavage sites in both methods of analysis. It was proposed that the enzyme recognises the narrower minor groove of these consecutive AT base pairs and cleaves DNA 3' to this feature.

Cisplatin Analogues with an Increased Interaction with DNA: Prospects for Therapy.

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.