COMPARISON OF REPAIR ACTIVITY IN DIFFERENT GENOMIC REGIONS

We have developed a quantitative technique to determine repair activit y at defined genomic regions. Cells were treated with hydroxyurea to i nhibit the replicative DNA synthesis and were incubated with 5-bromode oxyuridine (BrdUrd) to label the regions undergoing repair. In the cou rse of the labelling, the regions that were more actively repaired wou ld incorporate more BrdUrd than the regions that were less actively re paired. Thus the kinetics of BrdUrd incorporation in the different seq uences would reflect the kinetics of reparation of the respective regi ons. The total BrdUrd-containing, repaired DNA was isolated by immunop recipitation with anti-BrdUrd antibody, and after controlled sonicatio n, it was used as a template in quantitative PCR in which the amount o f the product was directly proportional to the amount of template. Thi s approach was used to address the question whether DNA repair after U V irradiation occurs in an uniformly random manner, or with preference s for certain regions. We found that, in Ehrlich ascites tumor cells, the repair efficiency was higher at the 5' end of the mouse beta-globi n domain than in the rest of the domain.