Nuclear distribution of human DNA topoisomerase IIbeta: a nuclear targeting signal resides in the 116-residue C-terminal tail.
We have analyzed the subcellular distribution of the beta isoform of human topoisomerase II using both isoform-specific antisera and an epitope-tagging approach. Previous immunocytochemical studies have yielded differing results with one reporting this isoform to be predominantly nucleolar. Later studies seem to refute this finding, as do our results with isoform-specific antisera reported here. Epitope tagging minimizes potential complications arising from the use of anti-topoisomerase II antisera that may recognize epitopes that are modified or masked in vivo and could lead to misleading results in immunocytochemical studies. A second strength of this approach is that it allowed a comparison with similarly tagged control proteins (derived from the nucleolar transcription factor UBF) that were known to localize unambiguously to the cytoplasmic, nucleoplasmic, or nucleolar compartments. We report that the C-terminal domain of topoisomerase IIbeta fused to a beta-galactosidase tag localizes to the nucleus (but not the nucleolar compartment) and that this is indistinguishable from the localization of native topoisomerase IIbeta detected by isoform-specific antisera. Further analysis revealed that the nuclear localization determinant lies within the 116-residue C-terminal tail of human topoisomerase IIbeta.
Cowell
I G
IG
The Medical School, University of Newcastle, Newcastle-upon-Tyne, NE2 4HH, United Kingdom.
Willmore
E
E
Chalton
D
D
Marsh
K L
KL
Jazrawi
E
E
Fisher
L M
LM
Austin
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D019556:COS Cells; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D007536:Isomerism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D004268:DNA-Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D004250:DNA Topoisomerases, Type II