The Second Zinc-Finger Domain of Poly(ADP-Ribose) Polymerase Determines Specificity for Single-Stranded Breaks in DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 1990-04, Vol.87 (8), p.2990-2994
• Main Authors: Gradwohl, Gerard, De Murcia, Josiane Menissier, Molinete, Miguel, Simonin, Frederic, Koken, Marcel, Jan H. J. Hoeijmakers, de Murcia, Gilbert
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analytical, structural and metabolic biochemistry; Base Sequence; Biochemistry, Molecular Biology; Biological and medical sciences; Cell Line; Cellulose nitrate; Cloning, Molecular; Complementary DNA; DNA; DNA, Single-Stranded; DNA, Single-Stranded - metabolism; DNA-Binding Proteins; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Enzymes; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Life Sciences; Ligands; Metalloproteins; Metalloproteins - genetics; Metalloproteins - metabolism; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Oligonucleotides; Poly(ADP-ribose) Polymerases; Poly(ADP-ribose) Polymerases - genetics; Poly(ADP-ribose) Polymerases - metabolism; Polymerase chain reaction; Protein Conformation; Proteins; Structural Biology; Substrate Specificity; Transferases; Zinc; Zinc - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.8.2990

Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a zinc-binding protein that specifically binds to a DNA strand break in a zinc-dependent manner. We describe here the cloning and expression in Escherichia coli of a cDNA fragment encoding the two putative zinc fingers (FI and FII) domain of the human poly(ADP-ribose) polymerase. Using site-directed mutagenesis, we identified the amino acids involved in metal coordination and analyzed the consequence of altering the proposed zinc-finger structures on DNA binding. Disruption of the metal binding ability of the second zinc finger, FII, dramatically reduced target DNA binding. In contrast, when the postulated Zn(II) ligands of FI were mutated, the DNA binding activity was only slightly affected. DNase I protection studies showed that the FII is involved in the specific recognition of a DNA strand break. These results demonstrate that poly(ADP-ribose) polymerase contains a type of zinc finger that differs from previously recognized classes in terms of both structure and function.