DNA damage and repair provoked by
ethidium azide (EA) photoaffinity labeling in mouse
leukemia cells was studied by measuring sedimentation properties of nucleoids in neutral
sucrose gradients, and it was found that the strand opening step was faster than that which followed damage of cells by ultraviolet (UV) light. The two insults were compared at levels of damage which gave the same overall rates of repair synthesis in intact cells and which required the same length of time to complete repair, as judged by the restoration of supercoiling of the isolated nucleoids. In the case of UV, single-strand breaks in
DNA were detectable at 30 min, maximum at 2 h, and the superhelical properties restored at 21 h. With photoaffinity labeling, single-strand breaks were prominent immediately, even when photolabeling of cells was done on
ice, but restoration of
DNA supercoiling still required 21 h. Photolabeling of isolated nucleoids or isolated
viral DNA with EA failed to introduce
DNA strand breaks. However, it was discovered that photoaffinity labeling of
DNA with EA resulted in
alkali labile sites shown by single strand breaks produced on alkaline
sucrose sedimentation or by
alkali exposure followed by sedimentation on neutral
formamide gradients. These results suggest that the
drug attachment sites should be identifiable by the location of such single strand breaks.