scholarly journals Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II.

1992 ◽  
Vol 119 (5) ◽  
pp. 1023-1036 ◽  
Author(s):  
K Shiozaki ◽  
M Yanagida
Keyword(s):  
Fission Yeast ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Essential Role ◽  
T Antigen ◽  
Nuclear Localization Sequence ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Topo Ii ◽  
Terminal Domains

Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast.

scholarly journals A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II.

1991 ◽  
Vol 11 (12) ◽  
pp. 6093-6102 ◽  
Author(s):  
K Shiozaki ◽  
M Yanagida
Keyword(s):  
Amino Acids ◽  
Fission Yeast ◽  
Topoisomerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
The Core ◽  
Topo 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.

Ultrastructural localization of DNA topoisomerase II in oral squamous cell carcinoma utilizing immunogold EM

1992 ◽  
Vol 50 (1) ◽  
pp. 644-645
Author(s):  
M D'Andrea ◽  
P Farber ◽  
D Foglesong
Keyword(s):  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Essential Role ◽  
Ultrastructural Localization ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Dna Topoisomerases ◽  
Dividing Cells ◽  
Topo Ii

DNA topoisomerases are enzymes which insert transient breaks into duplex DNA and are important in DNA replication, transcription and recombination. There are two forms of the enzyme, Topoisomerase I which breaks and rejoins only one of the two strands of DNA and Topoisomerase II (Topo II) which breaks and rejoins both of the DNA duplex strands. The essential role of Topo II in cell proliferation is reflected in its abundance in dividing cells, including normal and neoplastic cells.

A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II

1991 ◽  
Vol 11 (12) ◽  
pp. 6093-6102
Author(s):  
K Shiozaki ◽  
M Yanagida
Keyword(s):  
Amino Acids ◽  
Fission Yeast ◽  
Topoisomerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
The Core ◽  
Topo 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.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

scholarly journals Rearrangements of the MLL gene in therapy-related acute myeloid leukemia in patients previously treated with agents targeting DNA- topoisomerase II

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3705-3711 ◽  
Author(s):  
HJ Super ◽  
NR McCabe ◽  
MJ Thirman ◽  
RA Larson ◽  
MM Le Beau ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Topoisomerase Ii ◽  
De Novo ◽  
Chromosome Band ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mll Gene ◽  
Topo Ii ◽  
Acute Myeloid

Chromosome band 11q23 is frequently involved in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) de novo, as well as in myelodysplastic syndromes (MDS) and lymphoma. Five percent to 15% of patients treated with chemotherapy for a primary neoplasm develop therapy-related AML (t-AML) that may show rearrangements, usually translocations involving band 11q23 or, less often, 21q22. These leukemias develop after a relatively short latent period and often follow the use of drugs that inhibit the activity of DNA-topoisomerase II (topo II). We previously identified a gene, MLL (myeloid-lymphoid leukemia or mixed-lineage leukemia), at 11q23 that is involved in the de novo leukemias. We have studied 17 patients with t-MDS/t-AML, 12 of whom had cytogenetically detectable 11q23 rearrangements. Ten of the 12 t-AML patients had received topo II inhibitors and 9 of these, all with balanced translocations of 11q23, had MLL rearrangements on Southern blot analysis. None of the patients who had not received topo II inhibitors showed an MLL rearrangement. Of the 5 patients lacking 11q23 rearrangements, some of whom had monoblastic features, none had an MLL rearrangement, although 4 had received topo II inhibitors. Our study indicates that the MLL gene rearrangements are similar both in AML that develops de novo and in t-AML. The association of exposure to topo II- reactive chemotherapy with 11q23 rearrangements involving the MLL gene in t-AML suggests that topo II may play a role in the aberrant recombination events that occur in this region both in AML de novo and in t-AML.

Immunocytometric detection of the α-and β-isoforms of DNA topoisomerase II (topo II) in human ovarian cancers

1993 ◽  
Vol 29 ◽  
pp. S136
Author(s):  
C. Oliani ◽  
E. Prosperi ◽  
P. Biondani ◽  
C. Griso ◽  
F. Pavanel ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Ovarian Cancers ◽  
Topo Ii
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