Oxidative repair of a thymine dimer in DNA from a distance by a covalently linked organic intercalator

A thymine cyclobutane dimer, site-specifically incorporated in a DNA duplex, is shown to be repaired upon photoexcitation (at 380 nm) of a naphthalene diimide intercalator (NDI), either bound noncovalently to the duplex or covalently appended to the C4 amine of a methylated cytosine base well separated from the thymine dimer. The repair of the thymine dimer is triggered by photooxidation either directly or by DNA-mediated charge transport over a distance of similar to 22 Angstrom, the separation between NDI and the cyclobutane ring. Photooxidative repair with covalently and noncovalently bound NDI is demonstrated using HPLC under denaturing conditions, where the loss of the thymine dimer-containing strand and the formation of the repaired strand are monitored directly, as well as using a novel gel electrophoretic assay. In this assay, two strands of oligonucleotides containing 5'- and 3'-terminal thymidines are first ligated photochemically to yield thymine dimers, and repair is then assayed by monitoring the reversal of the photoligation by intercalators bound either noncovalently or at a distance. Although both NDI and a rhodium intercalator were seen to reverse the photoligation, several anthraquinones and ethidium were unable to promote repair upon irradiation at 350 nm. This photoligation reversal assay provides a rapid screen for thymine dimer repair. The oxidative repair of thymine dimers in a DNA duplex from a distance appears now to be a general phenomenon and requires consideration in developing mechanisms for DNA-mediated charge transport.