The second zinc-finger domain of poly(ADP-ribose) polymerase determines specificity for single-stranded breaks in DNA.
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.
Gradwohl
G
G
Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Laboratoire de Biochimie II, Strasbourg, France.
Ménissier de Murcia
J M
JM
Molinete
M
M
Simonin
F
F
Koken
M
M
Hoeijmakers
J H
JH
de Murcia
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008667:Metalloproteins; D015345:Oligonucleotide Probes; D011065:Poly(ADP-ribose) Polymerases; D015032:Zinc