A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II.
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.
Shiozaki
K
K
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Yanagida
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005816:Genetic Complementation Test; D006238:Haploidy; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010206:Papain / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D016415:Sequence Alignment
D004271:DNA, Fungal; D010450:Endopeptidases; D010206:Papain; D004250:DNA Topoisomerases, Type II