Two separate conserved domains of eukaryotic DNA topoisomerase I bind to each other and reconstitute enzymatic activity.
The two-hybrid system was used to identify proteins that interact with the central conserved domain of Saccharomyces cerevisiae DNA topoisomerase I. Several different C-terminal domain-containing fragments of topoisomerase I, none of which overlapped with the central domain, were identified as specific interacting polypeptides. Coexpression of these two domains in yeast partially complemented the growth defects of top1-top2ts and top1-hpr1 mutants. Moreover, an in vitro assay showed that some topoisomerase I enzymatic activity was restored to these mutants. The results demonstrate that the central domain of topoisomerase I interacts with the C-terminal domain of the protein and that these two domains reconstitute enzymatic activity in vivo, even when expressed as separate polypeptides.
Park
H
H
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
Sternglanz
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000737:chemistry / Q000201:enzymology; D005816:Genetic Complementation Test; D006822:Hybrid Cells / Q000201:enzymology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics
D010446:Peptide Fragments; D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I