Comparison of repair activity in different genomic regions.

Abstract

We have developed a quantitative technique to determine repair activity at defined genomic regions. Cells were treated with hydroxyurea to inhibit the replicative DNA synthesis and were incubated with 5-bromodeoxyuridine (BrdUrd) to label the regions undergoing repair. In the course of the labelling, the regions that were more actively repaired would incorporate more BrdUrd than the regions that were less actively repaired. Thus the kinetics of BrdUrd incorporation in the different sequences would reflect the kinetics of reparation of the respective regions. The total BrdUrd-containing, repaired DNA was isolated by immunoprecipitation with anti-BrdUrd antibody, and after controlled sonication, it was used as a template in quantitative PCR in which the amount of the product was directly proportional to the amount of template. This approach was used to address the question whether DNA repair after UV irradiation occurs in an uniformly random manner, or with preferences for certain regions. We found that, in Ehrlich ascites tumor cells, the repair efficiency was higher at the 5' end of the mouse beta-globin domain than in the rest of the domain.