TY - JOUR
T1 - The second zinc-finger domain of poly(ADP-ribose) polymerase determines specificity for single-stranded breaks in DNA
AU - Gradwohl, G.
AU - Menissier De Murcia, J.
AU - Molinete, M.
AU - Simonin, F.
AU - Koken, M.
AU - Hoeijmakers, J. H.J.
AU - De Murcia, G.
PY - 1990
Y1 - 1990
N2 - 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 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.
AB - 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 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.
KW - DNA-binding protein
KW - DNase I protection assay
KW - site-directed mutagenesis
KW - zinc-binding protein
UR - http://www.scopus.com/inward/record.url?scp=0025232413&partnerID=8YFLogxK
U2 - 10.1073/pnas.87.8.2990
DO - 10.1073/pnas.87.8.2990
M3 - Article
C2 - 2109322
AN - SCOPUS:0025232413
SN - 0027-8424
VL - 87
SP - 2990
EP - 2994
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 8
ER -