Visualization of chromatin events associated with repair of ultraviolet light-induced damage by premature chromosome condensation.
The purpose of this study was to characterize a system with which to study chromatin events associated with the repair of u.v. light-induced damage. Quiescent normal human fibroblasts were irradiated with u.v. and the ensuing chromatin events were visualized by inducing premature chromosome condensation in the treated cells. Treatment with u.v. induced the following two types of chromatin changes reflected in the morphology of G1 premature condensed chromosomes (PCC): (i) a generalized elongation of the G1 PCC and (ii) regions of localized elongation or gaps. The degree of chromatin change was dose dependent and could be seen immediately after irradiation. The generalized elongation process continued to increase for 24 h after irradiation, suggesting it represented a cellular reaction to the u.v.-induced damage, rather than a direct physical distortion. The localized decondensation reaction was associated with the site of unscheduled DNA synthesis. Posttreatment incubation of cells in the presence of cytosine arabinoside and hydroxyurea resulted in an accumulation of gaps. The inhibitor novobiocin predominantly inhibited the formation of gap regions, suggesting that a topoisomerase-like reaction might be important in their formation. The presence of cycloheximide after u.v. irradiation had no effect on the chromatin changes, suggesting that no new protein synthesis is required for these chromatin processes associated with repair. These results suggest that the PCC technique is useful in elucidating chromatin changes associated with DNA repair after u.v. treatment and can be used to elucidate chromatin events associated with the repair of other DNA-damaging agents.