Role of the DNA ligase III zinc finger in polynucleotide binding and ligation.
Mammalian DNA ligase III exists as two distinct isoforms denoted alpha and beta. Both forms possess a motif that is homologous to the putative zinc finger present in poly(ADP-ribose) polymerase. Here, the role of this motif in the binding and ligation of nicked DNA and RNA substrates in vitro has been examined in both isoforms. Disruption of the putative zinc finger did not affect DNA ligase III activity on nicked DNA duplex, nor did it abolish DNA ligase III-alpha activity during DNA base excision repair in a cell-free assay. In contrast, disruption of this motif reduced 3-fold the activity of both DNA ligase III isoforms on nicked RNA present in RNA/DNA homopolymers. Furthermore, whereas disruption of the motif did not prevent binding of DNA ligase III to nicked DNA duplex, binding to nicked RNA homopolymers was reduced approximately 10-fold. These results suggest that the putative zinc finger does not stimulate DNA ligase III activity on simple nicked DNA substrates, but indicate that this motif can target the binding and activity of DNA ligase III to nicked RNA homopolymer. The implications of these results to the cellular role of the putative zinc finger are discussed.
School of Biological Sciences, G.38 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
R M
RM
Taylor
J
J
Whitehouse
E
E
Cappelli
G
G
Frosina
K W
KW
Caldecott
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000075223:Poly-ADP-Ribose Binding Proteins; D011119:Polynucleotides / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins; D016335:Zinc Fingers / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011119:Polynucleotides; D011994:Recombinant Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins; D012313:RNA; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus