The effect of ionising radiation on DNA and its constituents : an EPR study

• Main Author: Malone, Mark E
• Format: Dissertation
• Language: English
• Published: University of Leicester 1993

This thesis was concerned with the effects of ?-radiation and UV-photoionisation on DNA an its constituents at 77 K. In particular, the work has concentrated on the radical-anion and radical-cation centres, with a view to obtaining a better understanding of the so-called direct mechanism of radiation damage. The ?-irradiation of DNA and its constituents in aqueous LiCl glasses suppresses radical cation formation and gives rise to well resolved radical anion spectra which can be readily studied by EPR spectroscopy. The nucleotides of adenine, cytosine, guanine and cytosine all yield base centred ?-radical anions, which are thought to undergo reversible deprotonation to form neutrally charged radicals. Electron competition studies in DNA, duplex polynucleotides, dinucleotides, and mononucleotide mixtures indicate that the electron affinities of the four bases are of the order C > T >> A ? G. However, the DNA radical anion (DNA. ), has a well resolved doublet spectrum which is indistinguishable from those of T.- and C.- in duplex systems. Consequently it is difficult to judge the proportions of C.- and T.- in these glasses. An alternative method to evaluate the proportions of C.- and T.- in irradiated DNA involves the annealing of frozen aqueous DNA in order to convert the T.- centres into TH. radicals, in which a hydrogen atom is added at C6. These results imply that ca 36 % of the radical anion centres in frozen aqueous DNA are due to T.- centres. The addition of LiCl, NaCl, LiBr and NaClO4 salts to DNA, cause a large increase in the yields of DNA radicals (mainly due to DNA.- centres). However, despite the increase in the radical yields, very little change in the yields of strand breaks are induced by these salts. The results are discussed in terms of an increase in the effective target volume and the reactivities of the salt radicals Cl2.-, Br.- and O.-. Perchlorate glasses and frozen aqueous matrices were employed to investigate the photoionisation of DNA and its constituents at 77 K. The perchlorate glasses scavenge the photoejected electrons and enables the isolation of the organic electron loss centres. In this manner, a number of electron loss radicals were identified. Thymine and cytosine yield N1 deprotonated ?-radical cations with spin centred at C5 and Nl. The radical cations of Nl substituted thymine derivatives (e.g. thymidine), deprotonate at the C5 methyl group to form the TCH2. radical. The nucleosides and nucleotides of cytosine give rise to sugar radicals which are thought to arise from single or multistep hydrogen shifts. A tentative assignment of these radicals has been made. The nucleotides of adenine and guanine give rise to well characterised ?-radical cations which are reversibly deprotonated at the exocyclic amino groups. Photoionisation of mononucleotides mixtures, dinucleotides and DNA indicate that the ease of photoionisation has the order C < T < A << G, and that guanine is the predominant electron loss centre in DNA. These results are discussed in terms of photophysical factors such as excited state lifetimes and ?isc, as well as energy and hole transfer.