Zinc-binding and protein-protein interactions mediated by the polyomavirus large T antigen zinc finger.
Polyomavirus large tumor antigen (LT) contains a potential C2H2 zinc binding element between residues 452 and 472. LT also contains a third histidine in this region, conserved among the polyomavirus LTs. Synthetic peptides of this region bound a single atom of zinc, as determined by spectroscopic analysis. Blotting experiments also showed that fusion proteins containing the element, as well as full-length LT, bound 65Zn. Polyomavirus middle T and small T antigens also bound zinc in the blotting assay. Site-directed mutagenesis showed the importance of this element in LT. Point mutations in four of the conserved residues (C-452, C-455, H-465, and H-469) blocked the ability of LT to function in viral DNA replication, while mutation of H-472-->L decreased replication to 1/30th that of the wild type. Point mutations in intervening residues tested had little effect on replication. Mutants resulting from mutations in the conserved cysteine or histidine residues retained the ability to bind origin DNA. However, they did show a defect in self-association. Because double-hexamer formation is involved in DNA replication, this deficiency is sufficient to explain the defect in replication. Mutants created by point mutations of the coordinating residues were also deficient in replication-associated phosphorylations.
Rose
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PE
Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Schaffhausen
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BS
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011120:Polyomavirus / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology*
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D015032:Zinc