Studies of the metal binding properties and DNA recognition mode of the unusual zinc fingers in poly(ADP-ribose) Polymerase-1 and the investigation of its interaction with apoptosis inducing factor (AIF)
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Poly(ADP-ribosyl)ation, a covalent modification of proteins catalyzed by poly(ADP-ribose) polymerases (PARPs), plays a crucial role in regulating DNA repair, DNA replication, and cell death. Poly(ADP-ribose) Polymerase-1 (PARP-1) is a nuclear zinc-finger DNA-binding protein that is the most extensively studied member of the PARP family. The activation of PARP-1 depends on the N-terminal DNA-binding domain, which consists of two unusually long zinc finger-like motifs (termed FI and FII) of the form CX₂CX₂₈/₃₀HX₂C and a newly discovered zinc-ribbon motif (FIII). Though zinc is indispensible for PARP-1 activity, the metal binding affinities of the unusual zinc fingers of PARP-1 is not yet known. In this dissertation, the second zinc finger of PARP-1 was used as a model peptide to study the binding properties of several divalent metal ions (Co²⁺, Cd²⁺, Zn²⁺, and Pb²⁺). Metal-induced protein folding was investigated by circular dichroism, and the effects of the metal ions on PARP-1 activity were investigated by poly(ADP-ribosyl)ation activity assays. This study represents the first detailed biochemical characterization of the PARP zinc fingers. The functional role of each zinc finger in DNA damage recognition is critical for understanding how PARP-1 is involved in DNA repair. Thus, we constructed a series of PARP-1 zinc finger variant proteins and investigated their DNA binding properties and their effects on PARP activity. Using a combination of southwestern blotting and activity assays, we demonstrated that FII is more important for DNA binding, while FI and FIII seem to facilitate PARP activity. The DNA sequence-independent binding properties of PARP-1 were further characterized using DNA probes bearing defined secondary structures. Together, our results indicate that the zinc fingers help position the enzyme at specific DNA damage sites, and also help to activate the catalytic domain upon DNA binding. PARP-1 is involved in caspase-independent apoptosis, and the translocation of apoptosis inducing factor (AIF) out of the mitochondrial matrix has been shown to require PARP-1 activity. However, it is not readily apparent how the catalytic activity of PARP-1 (a nuclear protein) triggers the release of AIF from the mitochondrial matrix. In an attempt to understand the relationship between PARP-1 activity and caspase-independent apoptosis, we demonstrate here that AIF is an in vitro protein substrate for PARP-1. The possible implications of this finding will be discussed.