Repair Pattern in the β‐Globin Gene Cluster of Human Fibroblasts after Ultraviolet Irradiation

Abstract

We have developed a novel technique to determine repair of structurally different DNA lesions. It was used to address the question of whether DNA repair in the absence of transcription occurs in a uniformly random manner or with preferences for certain regions. Human fibroblasts were exposed to ultraviolet light (3-10 J/m2) and treated with 7.5 mM hydroxyurea to inhibit replicative DNA synthesis. During the first hours after irradiation cells were treated with 5-bromodeoxyuridine to label the regions undergoing repair, with the presumption that the regions that have been more efficiently repaired would incorporate more of the nucleoside. A 155-kb DNA sequence containing the entire human beta-globin domain was reconstructed using sequences deposited in the EMBL gene bank. Twelve uniformly long single-copy RNA probes spanning the beta-globin cluster were synthesised in vitro and immobilized on microtiter plates. They were hybridized with DNA from the irradiated cells. The amount of 5-bromodeoxyuridine, incorporated as a result of repair in the DNA fractions hybridized to the different RNA probes, was determined immunochemically using antibody to this nucleoside. By this technique we registered increased repair efficiency in the zone of the permanent scaffold attachment region at the 5'-end of the beta-globin domain during the first hours after ultraviolet irradiation. This result was confirmed and by the more conventional T4 endonuclease V technique detecting the removal of cyclobutane pyrimidine dimers.