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

Abstract

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.

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

“…The inserted mitochondrial sequence corresponds to bp 11,851 to 12,097 in the hg19 human genome. The T7.GCGT.G10 sequence [60,61] is also based on pUC19 and consists of seven tandem repeats of a human telomeric sequence (5′-GGGTTA), a central 5′-GCGT repeating unit with adjacent sequence variants, and a sequence of ten consecutive G nucleotides. The J clone [60] contains a number of consecutive G nucleotides with different lengths.…”

“…The IR-induced DNA damage was generated by γ-irradiation from a sealed caesium-137 source that irradiated at a rate of 5.02 Gy/min at ambient atmosphere and temperature [60][61][62]. Experimental details of the three technique have been previously published for the LA/PS assay [60], the end-labelling technique [61] and the short-read Illumina next-generation genome-wide sequencing technique [62]. Purified DNA was utilised as the target for these three IR-induced DNA damage studies [60][61][62].…”

“…There are three techniques that have recently examined the DNA sequence preference of IR-induced DNA damage at nucleotide resolution using cutting-edge technology: the linear amplification/polymerase stop (LA/PS) assay [60], the end-labelling procedure [61], and Illumina next-generation genome-wide sequencing [62], as seen in Figure 1.…”

“…The end-labelling procedure mainly detects SSBs. A recent study observed that C nucleotides were the main site of IR-induced DNA damage [61].…”

“…The end-labelling procedure mainly detects SSBs. A recent study observed that C nucleotides were the main site of IR-induced DNA damage [61].In the Illumina next-generation genome-wide sequencing technique, a whole genome can be treated with IR and the precise sites of cleavage can be determined, as seen in Figure 1. In this technique, double-stranded linkers are enzymically added at the cleavage site, placed on the Illumina flowcell for sequencing, and tens of millions of sequencing reads can be obtained.…”