T-PM-Pol USE OF THE DYE HOECHST 33258 IN A MODIFICATION OF THE BROMODEOXYURIDINE PHOTOLYSIS TECHNIQUE FOR THE ANALYSIS OF DNA

T-PM-Pol USE OF THE DYE HOECHST 33258 IN A MODIFICATION OF THE BROMODEOXYURIDINE PHOTOLYSIS TECHNIQUE FOR THE ANALYSIS OF DNA REPAIR. B. S. Rosenstein* and R. B. Setlow (Intr. by M. Simon), Biology Dept., Brookhaven Nation¶l Laboratory, Upton, N.Y. 11973. A modification of the BrdU photolysis technique used in the measurement of the repair of damage in DNA has been developed employing the bisbenzimidazole dye Hoechst 33258. In the original technique 313 nm light is used to photolyze BrdU incorporated into parental DNA during repair. In the modified procedure cells are treated with 10 Ag/ml of the dye in PBS for 10 minutes and due to the broad absorption band (maximum at 356 nm) exhibited by chromatin bound dye, 365 nm light may then be used for the photolysis. The number of breaks in BrdU substituted DNA fre proportional to the extenf of substitution and to the dose. Approximately 2.1 X 10 breaks/BrdU residue per J/m are produced in fully substituted DNA from V79 cells by both 313 nm and 365 nm light with dye. The doses used to produce BrdU photolysis are therefore about the same for the two procedures but the modification has several advantages. 1) 365 nm light is not as strongly absorbed as 313 nm light by many chemical carcinogens and therefore some of the effects produced in unsubstituted, unrepaired DNA by 313 nm light may be avoided. 2) Irradiation times may be reduced because the output at 365 nm of a high pressure mercury arc lamp is about double that at 313 nm. 3) It is possible to use a low pressure "black light" as a source for photolysis because of the similarity of its emission spectrum and the absorption spectrum of the Hoechst dye when bound to chromatin. The latter advantage greatly simplifies the equipment necessary to carry out the BrdU photolysis procedure. (This work was supported by the Department of Energy and BSR is a Fellow in Cancer Research supported by Grant DRG-215-F of the Damon Runyon Walter Winchell Cancer Fund.) 1. Regan, J. D., R. B. Setlow and R. D. Ley. P. N. A. S. 68: 708, 1971.