Unusual Zn(II) Affinities of Zinc Fingers of Poly(ADP-ribose) Polymerase 1 (PARP-1) Nuclear Protein

Poly­(ADP-ribose) polymerase 1 (PARP-1) is a key eukaryotic enzyme, catalyzing the NAD+ dependent poly­(ADP-ribosyl)­ation of protein substrates, crucial for major DNA repair pathways, and involved in other fundamental cellular processes, such as transcription, cell cycle control, and apoptosis. Its ability to bind DNA depends on two CCHC zinc finger domains, in short, PARPzf1 and PARPzf2. Using spectroscopic methods and competitive titrations with Zn­(II), Co­(II), and Ni­(II) ions, we determined conditional dissociation constants for Zn­(II) complexes of PARPzf1 and PARPzf2 at pH 7.4 (HEPES buffer) as 26 ± 4 nM and 4 ± 1 pM, respectively. The former value indicates an extremely low affinity of PARPzf1 toward metal ions, meaning that under cellular conditions PARP1zf might be largely present in a “metal-free” state. This finding provides a clue to the high susceptibility of PARP-1 to oxidative stress but also raises questions regarding the activation of PARPzf1 under cellular conditions. We also determined conditional dissociation constants for Ni­(II) complexes of PARPzf1 and PARPzf2 under the same conditions as 0.78 ± 0.04 μM and 0.26 ± 0.05 nM, respectively.