X-ray-induced DNA damage spectrum in dilute aqueous solution: Selective protection by amino acid addition

DNA damage arises not only from the direct action of ionizing radiation but also from indirect action mediated by highly reactive hydroxyl radicals (^•OH). This study aimed to determine whether the protective or sensitizing effects of amino acids against X-ray-induced DNA damage are associated with the side-chain characteristics of these amino acids. Seven amino acids with distinct side-chain properties were investigated. The rate constants for their reactions with ^•OH were determined by electron pulse radiolysis. Highly purified, scavenger-free plasmid DNA (pUC18) was irradiated with X-rays in the presence of each amino acid, whose concentrations were adjusted to scavenge ∼14% of ^•OH. DNA strand breaks (SSBs and DSBs) were quantified by agarose gel electrophoresis. At the same time, oxidative base lesions and apurinic/apyrimidinic (AP) sites were detected as enzyme-sensitive sites (ESSs) using formamidopyrimidine-DNA glycosylase (Fpg), endonuclease III (Nth), and endonuclease IV (Nfo). Despite equivalent ^•OH scavenging capacities, the extent and spectrum of DNA damage varied markedly among the amino acids. Aspartic acid (Asp) and phenylalanine (Phe) exhibited the strongest overall protection, while glycine (Gly) showed the weakest protection, even with partial sensitization. Amino acids with highly hydrophilic or hydrophobic side chains tended to exhibit more substantial protective effects, although a clear correlation was not always observed for molecular weight or isoelectric point. Side-chain properties and potential chemical repair mechanisms are likely to contribute to the modulation of DNA damage. This work provides new mechanistic insights into amino acid-mediated DNA radioprotection, suggesting promising directions for molecular-level studies of amino-acid-DNA interactions.