| Autor: |
Adachi, Noritaka, Miyaike, Mitsuko, Kato, Satoshi, Kanamaru, Ryunosuke, Koyama, Hideki, Kikuchi, Akihiko |
| Zdroj: |
Nucleic Acids Research; August 1997, Vol. 25 Issue: 15 p3135-3135, 1p |
| Abstrakt: |
Mammalian cells express two genetically distinct isoforms of DNA topoisomerase II, designated topoisomerase IIα and topoisomerase IIβ. We have recently shown that mouse topoisomerase IIα can substitute for the yeast topoisomerase II enzyme and complement yeast top2 mutations. This functional complementation allowed functional analysis of the C-terminal domain (CTD) of mammalian topoisomerase II, where the amino acid sequences are divergent and speciesspecific, in contrast to the highly conserved N-terminal and central domains. Several C-terminal deletion mutants of mouse topoisomerase IIα were constructed and expressed in yeast top2 cells. We found that the CTD of topoisomerase IIα is dispensable for enzymatic activity in vitro but is required for nuclear localization in vivo. Interestingly, the CTD of topoisomerase IIβ was also able to function as a signal for nuclear targeting. We therefore examined whether the CTD alone is sufficient for nuclear localization in vivo. The C-terminal region was fused to GFP (green fluorescent protein) and expressed under the GAL1 promoter in yeast cells. As expected, GFP signal was exclusively detected in the nucleus, irrespective of the CTD derived from either topoisomerase IIα or IIβ. Surprisingly, when the upstream sequence of each CTD was added nuclear localization of the GFP signal was found to be cell cycle dependent: topoisomerase IIα-GFP was seen in the mitotic nucleus but was absent from the interphase nucleus, while topoisomerase IIβ-GFP was detected predominantly in the interphase nucleus and less in the mitotic nucleus. Our results suggest that the catalytically dispensable CTD of topoisomerase II is sufficient as a signal for nuclear localization and that yeast cells can distinguish between the two isoforms of mammalian topoisomerase II, localizing each protein properly. |
| Databáze: |
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