GzmC induces single-strand DNA nicking.

(A) Flow-TUNEL analysis was performed on YAC1 cells treated with perforin plus 1 μM rGzmB (○) or rGzmC (▿) for the times specified. Untreated YAC1 cells (■) and cells incubated with perforin (▵), rGzmB (∗), or rGzmC (⋄) alone were also analyzed for FITC-TUNEL positivity. (B) YAC1 target cells were untreated or treated with perforin, rGzmB, rGzmC, perforin plus rGzmB, or perforin plus rGzmC (1 μM). Genomic DNA was harvested after incubation at 37°C for 1 or 2 hours and was visualized on an ethidium bromide-stained 2% agarose gel. To estimate cell viability in the same experiment, the percentage of 7-AAD^lo target cells was analyzed by flow cytometry (values for each sample are shown below each line). (C) Genomic DNA from YAC1 target cells treated as in panel B were radiolabeled with ³²P-dATP by the Klenow fragment of DNA polymerase I. DNA fragments were separated with denaturing alkaline gel electrophoresis and autoradiographed.