Induction of photoproducts in synthetic polynucleotides by far and near ultraviolet radiation.

Abstract

AbstractSingle and double‐stranded polynucleotides of thymine and cytosine have been used to analyse the photoproducts produced by irradiation with far or near UV light. Reversed‐phase high performance liquid chromatography was used to detect and quantify cyclobutane dimers and Pyr(6–4)Pyo adducts produced by 254 nm. At 320 nm 10‐times less Thy(5–6)Thy dimers and one half the number of Cyt(5–6)Cyt dimers were induced; no Pyr(6–4)Pyo adducts were evident.HPLC has recently been applied to the isolation and characterization of various nucleic acid substituents and their UV‐photoproducts. The relative retention times of pyrimidines and their UV photoproducts on HPLC reflect differences in the hydrophobicity of the compounds being separated. Hence, the more hydrophobic (less polar) a compound is, the greater its capacity to bind to the column and the greater its retention time; for T o T, C<>T and C<>C dimers this difference may result from variations in the number of methyl moieties (Cadet et al., 1983). The retention time of the compound also depends on its stereochemistry. Separation of the four stereoisomers of T<>T by HPLC shows that compounds which are molecular equivalents can be more or less accessible to the non‐polar stationary phase depending on their conformation (Cadet, 1980). The relatively long retention times of the bipyrimidine photoadducts suggest a structural configuration which allows greater access to the hydrophobic moieties of the molecule.It is intriguing to consider that this difference in conformation may also be reflected in DNA–protein interactions such as binding by UV‐endonucleases or antibodies directed against UV photodamage. Proteins can be used as sensitive probes for photoproduct induction and repair but an accurate evaluation of their specificity is required. It was the intent of this paper not only to compare the induction parameters for various dimers in pyrimidine homo‐polymers but to provide controlled substrates which can be used to define the relative binding efficiencies of repair enzymes and antibodies for different types of photoproducts.