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. 2016 Aug 16;44(20):9771–9783. doi: 10.1093/nar/gkw710

Figure 6.

Figure 6.

Inefficient intermolecular activation of PARP-1. Mixtures of PARP-1 fragments and mutants were analyzed in the presence of DNA damage to observe the efficiency of intermolecular activation. (A) Both WT and W318R versions of the acrylodan-labeled PARP-1 mutant K700C (0.5 μM) were mixed with unlabelled PARP-1/WT and mutants (0.5 μM) in the presence of dsDNA (1 μM). Detection was determined from the emission ratio (λ460/λ535) that reveals the extent of HD destabilization. (B) A schematic representation of intramolecular and intermolecular activation mechanisms. (C) The acrylodan-labeled PARP-1/WT and W318R mutant (0.5 μM) were mixed with dsDNA (1 μM) in the presence and absence of unlabeled PARP-1/WT (0.5 μM). Acrylodan-labeled W318R mutant was also mixed with unlabeled PARP-1 mutants. Mixtures were reacted with NAD+ for the specified amount of time and then resolved on 10% SDS-PAGE and visualized under UV light so that only the fluorescent acrylodan-labeled PARP-1 is visible. Data points were conducted in triplicates ± S.D.