Disease relevance of TOP2

• The gene TOP2 encoding yeast topoisomerase II has been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector, lambda gt11 [1].
• The mutant with partial resistance to etoposide and fluoroquinolones has an amino acid change at position 738 of TOP2, which is three amino acids from the site homologous to Ser83 of E. coli gyrA, an amino acid which had previously been shown to be an important target for resistance to quinolones in bacteria [2].
• Interestingly, Ser741-->Trp in the yeast TOP2 also confers hypersensitivity to etoposide [3].
• Suramin is an inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells [4].
• At the restrictive temperature, top2 strains show multiple abnormalities, including an inability to complete mitotic and meiotic division owing to a defect in chromosome segregation, and hyper-recombination within the repetitive rDNA gene cluster [5].

High impact information on TOP2

• This phenotype is similar to that described in TOP2 mutants in yeast [6].
• To understand better the similarities and differences between meiosis and mitosis, we examined the meiotic role of DNA topoisomerase II, an enzyme that is required mitotically to disentangle sister chromatids at the time of chromosome segregation [7].
• Segregation of recombined chromosomes in meiosis I requires DNA topoisomerase II [7].
• When cold-sensitive top2 mutants are induced to sporulate at the restrictive temperature, they undergo premeiotic DNA synthesis and commitment to meiotic levels of recombination but fail to complete the first meiotic nuclear division [7].
• Expression of a plasmid-borne TOP1 or TOP2 gene in the strain leads to the integation of the extrachromosomal rDNA rings back into the chromosomal rDNA cluster [8].

Chemical compound and disease context of TOP2

• Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II [9].
• Aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells [10].

Biological context of TOP2

• The high frequency of mitotic recombination within the rDNA cluster in topoisomerase mutants shows that both TOP1 and TOP2 are required for suppression of recombination in this region of the genome [11].
• Candida albicans topoisomerase II, encoded by the TOP2 gene, mediates chromosome segregation by a double-strand DNA break mechanism and is a potential target for anti-fungal therapy [12].
• It was used to construct a plasmid in which TOP2 expresses a recombinant enzyme (residues 57-1461 of C. albicans topoisomerase II fused to the first five residues of Saccharomyces cerevisiae topoisomerase II) under the control of a galactose-inducible promoter [12].
• In human cells, atypical drug resistance was previously identified with reduced catalytic activity or nuclear localization efficiency of DNA topoisomerase II alpha (TOP2 alpha) [13].
• Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centi-Morgans to the centromere proximal side of MET4 [14].

Anatomical context of TOP2

• We also have constructed mutations in human TOP2 based on previously identified mutations from a human cell line selected for resistance to teniposide [15].
• The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis [16].
• Mutations in DNA topoisomerase II are often correlated with drug-resistance in tumor cell lines [17].
• Several cDNA clones encoding mouse DNA topoisomerase II were obtained from a mouse spermatocyte cDNA library and the entire coding sequence of the gene was determined [18].

Associations of TOP2 with chemical compounds

• A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene [19].
• Purified recombinant proteins were 8- to 12-fold more resistant to etoposide and doxorubicin than wild type TOP2 alpha, and 2-fold more resistant to amsacrine, as measured by accumulation of cleavable DNA [13].
• Moreover, at concentrations as low as 5 microM, CP-115,953 was cytotoxic to yeast cells that carried wild type topoisomerase II (TOP2+) [20].
• Galactose-dependent expression of the cloned rat topo II alpha cDNA complemented a yeast top2ts mutation, as well as a deletion mutation at the yeast TOP2 locus [21].
• Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands [22].

Regulatory relationships of TOP2

• TOP2 suppresses mini-chromosome loss in pds5 mutants indicating that it rescues a chromosome segregation defect [23].
• The mutagenized TOP2 gene we have used is under the control of the yeast DED1 promoter; this overexpression of TOP2 is designed to avoid isolating mutants that are drug resistant solely because the mutated topoisomerase II has low enzymatic activity [2].
• METHODS: A drug permeable yeast strain (JN394 top2-4) was transformed using a shuttle vector containing either human top2alpha, human top2alpha or yeast top2 under the control of a GAL1 promoter [24].

Other interactions of TOP2

• Strains with a null mutation in the TOP1 gene (encoding topoisomerase I) or a ts mutation in the TOP2 gene (encoding topoisomerase II) grown at a semipermissive temperature show 50- to 200-fold higher frequencies of mitotic recombination in rDNA relative to TOP+ controls [11].
• I mapped MKT1 near TOP2 and isolated the gene by chromosome walking from TOP2 [25].
• The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2 [26].
• TOP2 suppression is specific for pds5 mutants as it does not suppress mutants in the cohesin complex [23].
• The activities of the three nuclear DNA topoisomerases (Top1, Top2, and Top3) in the yeast Saccharomyces cerevisiae were examined for their influence upon the nucleotide excision repair (NER) of DNA damage induced by ultraviolet (UV) irradiation [27].

Analytical, diagnostic and therapeutic context of TOP2

• Northern blot analysis of TOP2 RNA isolated from a wild-type strain and from an rna2 mutant shows the RNA to be 4.5 kb long in both cases, thus indicating that the TOP2 gene has no large introns [14].
• Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA [28].
• Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast [12].
• Fluorescence and electron microscopy reveal that top2 mutants exhibit wild-type levels of meiotic chromosome condensation and form morphologically normal synaptonemal complex but are delayed in the exit from pachytene [29].
• Study of yeast DNA topoisomerase II and its truncation derivatives by transmission electron microscopy [30].

References