FIG. 6.
Effect of poly(ADP-ribosyl)ated PARP-1 on DNA joining by DNA ligase III. (A) Purified PARP-1 (100 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (50 nM) in the presence (+) or absence (−) of NAD. Subsequently, intact DNA ligase III (Lig III; 5 nM) or a truncated version lacking the zinc finger (ΔZf-Lig III; 5 nM) was added to the reaction mixture as indicated. After incubation for 10 min at 25°C, labeled oligonucleotides were separated by denaturing gel electrophoresis. The labeled substrate (30-mer) and ligated product (50-mer) were detected and quantitated by phosphorimager analysis. (B) The results of three independent DNA joining assays are shown graphically. White bars, full-length DNA ligase IIIβ; grey bars, truncated version of DNA ligase IIIβ lacking the zinc finger.