Given the right conditions, DNA and proteins can combine to form DNA-protein crosslinks and the early studies of Smith (1969) indicate that the most reactive amino acids for forming DNA-protein crosslinks (DPCs) are cysteine, tyrosine and phenylalanine. It has been suggested that formation of these crosslinks involves direct covalent bonding between protein amino acids and specific bases in DNA (Cress & Bowden, 1983). DNA-protein crosslinks can be produced by a wide range of chemicals and radiations which act via free radical formation.
These crosslinks are not efficiently repaired and may interfere with normal functions of the nuclear matrix, ultimately leading to cell death. Additional information concerning DNA-protein crosslinks is likely to be of considerable value in our understanding of the ageing process.
One likely site for DNA-protein crosslinking is between thymine and tyrosine residues. Others have demonstrated the formation of a thymine-tyrosine adduct in a gamma-irradiated equimolar solution of thymine and tyrosine (Dizdaroglu, 1984) and this product was characterised by Margolis et al (1988), and subsequently quantified by Simic & Dizdaroglu (1985). To undertake additional studies of this DNA-protein crosslinking we sought to confirm previous reports of the formation of the adduct and then to develop an assay for its quantification in biological systems. Precise quantification is best performed with the use of a stable isotope labelled internal standard and we therefore set about preparing some reference material and a suitable stable labelled isotopomer. Attempts to prepare the adduct by irradiation of an aqueous solution of both tyrosine and thymine proved unsuccessful. The adduct was formed at trace levels along with an abundance of other compounds generated by the ionising radiation. Irradiation was therefore not practical for the preparation of large (mg) quantities of material. The thymine-tyrosine adduct (3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine) was synthesised chemically from 5-hydroxy methyluracil and L-tyrosine. A d-2 labelled stable isotopomer was also prepared using the same approach by substituting d-2-L-tyrosine for native tyrosine. These syntheses provided analytically pure samples in good yield.
We have used both GCMS and LCMS together with the analytical standard, in a re-examination of the products formed by gamma-irradiation of an aqueous solution of tyrosine and thymine. Further, the synthetic products are now being used to develop an LCMS method (combining collision activated dissociation and select reaction monitoring) for the sensitive and specific quantification of the adduct in biological samples.