GzmA binds to PARP-1. (A) PARP-1 in cell lysates binds to S-AGzmA. Precleared HeLa cell lysates were incubated with S-AGzmA and antisera to PARP-1 or control antisera, and immune complexes were captured on protein G beads. GzmA binding was assayed by immunoblot probed with His-tag antibody. DNase treatment after PARP-1/GzmA incubation does not disrupt the association of S-AGzmA and PARP-1. (B) Recombinant PARP-1 binds S-AGzmA in vitro only in the presence of DNA. Recombinant PARP-1 attached to PARP-1 antibody–conjugated beads was incubated with S-AGzmA for 3 hours on ice in the presence or absence of 100-bp PCR product DNA. S-AGzmA was pulled down with PARP-1 only in the presence of DNA. However, if DNase I was added before extraction in SDS sample buffer, binding was not disrupted. (C) PARP-1 is not a component of the SET complex. Purified SET complex and K562 cell lysates were probed for PARP-1 and for the known SET complex components SET, pp32, and NM23-H.8 (D) GzmA disrupts the PARylation activity of PARP-1. Recombinant PARP-1 was preincubated with the indicated amount of GzmA at 37°C for 1 hour and then incubated with radiolabeled NAD+ before electrophoresis and autoradiography. The radiolabeled band corresponds to automodified PARP-1.
