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

Abstract

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.

Keywords:

• DNA sequence preference
• gamma radiation
• ionizing radiation
• linear amplification
• polymerase stop assay

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the University of New South Wales, Science Faculty Research Grant Scheme under Grant [2012-2018]. MEH was supported by an Australian Postgraduate Award.

Notes on contributors

Megan E. Hardie

Megan E. Hardie, BSc, is a postgraduate student in the School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.

Vincent Murray

Vincent Murray, PhD, is an Associate Professor in the School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.