Poly(ADP-ribose)polymerase: a novel finger protein

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Poly(ADP-ribose) polymerase (PARP) is a key enzyme involved in DNA repair.
• The role of metal ions, particularly zinc, in PARP function is not fully understood.
• Previous studies suggested the presence of zinc-binding motifs in PARP.

Purpose of the Study:

• To determine the stoichiometry of zinc binding to calf thymus poly(ADP-ribose) polymerase.
• To identify the specific domains responsible for zinc binding.
• To investigate the functional significance of zinc in PARP-DNA interactions.

Main Methods:

• Energy Dispersive X-ray fluorescence (EDXRF) was used to quantify zinc binding.
• Radioisotope 65Zn (II) was employed for western blot detection of zinc-binding proteins.
• Southwestern assays were performed to assess DNA binding activity after metal depletion and restoration.

Main Results:

• Calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule.
• Zinc binding sites are located in a 29 kDa N-terminal fragment, part of the DNA binding domain.
• Zinc is essential for the binding of this fragment to DNA, as demonstrated by Southwestern assays.

Conclusions:

• Zinc ions are integral components of calf thymus poly(ADP-ribose) polymerase, essential for its DNA binding capability.
• The N-terminal zinc-binding domain, containing putative zinc finger motifs, likely plays a critical role in recognizing DNA strand breaks.
• These findings suggest that zinc fingers in PARP are involved in enzyme activation through DNA damage recognition.