Topoisomerase I from chicken erythrocytes: purification, characterization, and detection by a deoxyribonucleic acid binding assay.
We have purified a deoxyribonucleic acid topoisomerase to near homogeneity from the nuclei of mature chicken erythrocytes. The enzyme relaxes supercoiled DNA in the absence of ATP or Mg2+. It is unable to resolve topologically knotted circular duplex DNA. These properties resemble those of type I eukaryotic topoisomerases capable of breaking and rejoining one strand of duplex DNA at a time. The sedimentation value of the protein is 4.4 S. The molecular weight of the reduced, denatured protein is 100K. After elution from sodium dodecyl sulfate (NaDodSO4) gels and renaturation, topoisomerase activity is found in the band at 100K and in minor bands at 95K, 78K, and 73K. The minor bands are likely to be proteolytic fragments since the Mr 100K protein is cleaved by trypsin to fragments of similar or even smaller size with retention of activity. At KCl concentrations suboptimal for the 100K form, the trypsin cleaved form is severalfold more active than the 100K form. Single-stranded DNA, but not duplex DNA or RNA, inhibits DNA relaxing activity, presumably by forming a covalent complex at the enzyme active site. Preincubation of the enzyme with single-stranded DNA leads to the depletion, in NaDodSO4-polyacrylamide gels, of protein bands corresponding to the 100K topoisomerase, its putative proteolytic fragments, and its tryptic fragments. The reaction which leads to band depletion requires active topoisomerase and conditions where single-stranded DNA inhibits relaxing activity. The band depletion technique provides a convenient assay for the polynucleotide binding activity of topoisomerases and possibly other proteins. The function of the enzyme in the inactive nuclei of mature chicken erythrocytes is unclear. The estimated content of chicken erythrocyte topoisomerase per unit DNA is comparable to that in nuclei active in replication and transcription.
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000097:blood* / Q000302:isolation & purification; D004912:Erythrocytes / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding
D004247:DNA; D004264:DNA Topoisomerases, Type I