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Updated: Aug 3, 2026
Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast
Published on: February 20, 2017
A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II
1Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Abstract:
We purified fission yeast DNA topoisomerase II (topo II) to apparent homogeneity. It consists of a single 165-kDa polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, upon treatment with a bifunctional reagent, doubles its molecular weight. Limited proteolysis of intact topo II by papain produces a 125-kDa core, which lacks the N-terminal 75 and the C-terminal approximately 260 amino acids but still contains regions similar to those of bacterial or phage T4 topo II subunits. The core retains relaxing and unknotting activities. Further digestion inactivates the core, cleaving it at the middle of the GyrB-like domain and at the beginning of the GyrA-like domain. Therefore, papain appears to cleave spatially distinct subdomains of topo II. We made top2 mutant genes deleted of the C-terminal 286 or N-terminal 74 amino acids, which can substitute for the wild-type top2+ gene in mitosis and meiosis. However, a mutant containing deletions of both termini cannot rescue the top2 null mutant, despite the fact that the product is enzymatically active. Therefore, the top2 product of the doubly truncated gene may not fulfill all of the in vivo requirements for top2+ function.
Insights
Fission yeast DNA topoisomerase II (topo II) was purified and characterized. Truncated topo II mutants retained enzymatic activity but failed in vivo, suggesting essential functions beyond catalysis.
Area of Science:
- Molecular Biology
- Enzymology
- Yeast Genetics
Background:
- DNA topoisomerase II (topo II) is crucial for managing DNA topology in all organisms.
- Understanding the structure-function relationship of topo II is key to comprehending its essential cellular roles.
Purpose of the Study:
- To purify and biochemically characterize fission yeast DNA topoisomerase II.
- To investigate the functional significance of different domains of topo II in vivo using genetic approaches.
Main Methods:
- Purification of fission yeast DNA topoisomerase II to homogeneity.
- Limited proteolysis with papain to identify functional domains.
- Construction and analysis of top2 mutant genes with deletions at N- and C-termini.
Main Results:
- Purified topo II is a 165-kDa polypeptide that forms a dimer upon crosslinking.
- A 125-kDa core fragment retaining relaxing and unknotting activity was generated by papain digestion.
- Mutants lacking either N- or C-terminal regions could complement top2 null mutants, but a double deletion mutant could not, despite retaining enzymatic activity.
Conclusions:
- Papain cleavage sites indicate spatially distinct subdomains within fission yeast topo II.
- The N- and C-terminal regions of topo II are essential for its in vivo function, independent of its catalytic activity.
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