Figure 2.

XK469-induced protein-DNA crosslinks to cellular and SV40 DNA. (A) Replicating SV40 genomes were pulse-labeled with ³H-dT for 30 min, and the drugs were added 15 min after the start of labeling. Labeling medium with drug was drawn off and the cells were lysed with Hirt lysing fluid. The Hirt supernatant, containing pulse-labeled SV40 DNA, was assayed for protein-SV40 DNA crosslinks. ○, R-isomer; ▵, S-isomer; ▾ R-isomer, proteinase K digested before assay; ⋄, S-isomer, proteinase K digested before assay. Error bars are ±SD derived from four points. (B) Reversal of S(−)XK469-induced protein-DNA crosslinks upon removal of the drug. SV40-infected CV-1 cells were labeled with ³H-dT beginning at t = −30 min. At t = −15 min, the labeling medium in all samples was made 1 mM in S(−)XK469. At t = 0, the labeling medium with XK469 in one set of samples was replaced with labeling medium without drug. At t = 2, 10, and 20 min, samples were harvested by removal of labeling medium and addition of Hirt lysing fluid. SV40 DNA was selectively extracted and measured for protein-DNA crosslinks (see Materials and Methods). (C) Camptothecin-induced topoisomerase I-cellular DNA crosslinks in drug-sensitive parental CV-1 cells (●) and in CPTCV10c22 cells (○) resistant to 1.5 μM camptothecin (CPT-r). (D) S(−)XK469-induced protein-DNA crosslinks in drug-sensitive parental CV-1 cells (●) and in CPTCV10c22 cells (○). (E) m-AMSA-induced topoisomerase II-DNA crosslinks in drug-sensitive parental CV-1 cells (●) and in AMCV1 cells (○) resistant to 3 μM m-AMSA. (F) S(−)XK469-induced protein-DNA crosslinks in parental CV-1 cells (●) and in AMCV1 (○) cells.