A Mutation in Yeast Topoisomerase II That Confers Hypersensitivity to Multiple Classes of Topoisomerase II Poisons

Jiaowang Dong,Jerrylaine Walker,John L Nitiss

doi:10.1074/jbc.275.11.7980

Jiaowang Dong, Jerrylaine Walker + Show 1 more

Open Access

https://doi.org/10.1074/jbc.275.11.7980

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Abstract

A mutation was constructed in the CAP homology domain of yeast topoisomerase II that resulted in hypersensitivity to the intercalating agent N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and the fluoroquinolone 6, 8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxyli c acid, but not to etoposide. This mutation, which changes threonine at position 744 to proline, also confers hypersensitivity to anti-bacterial fluoroquinolones. The purified T744P mutant protein had wild type enzymatic activity in the absence of drugs, and no alteration in drug-independent DNA cleavage. Enhanced DNA cleavage in the presence of N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and fluoroquinolones was observed, in agreement with the results observed in vivo. DNA cleavage was also seen in the presence of norfloxacin and oxolinic acid, two quinolones that are inactive against eukaryotic topoisomerase II. The hypersensitivity was not associated with heat-stable covalent complexes, as was seen in another drug-hypersensitive mutant. Molecular modeling suggests that the mutation in the CAP homology domain may displace amino acids that play important roles in catalysis by topoisomerase II and may explain the drug-hypersensitive phenotype.

Highlights

In addition to their numerous biological functions, topoisomerases are important targets for both anti-bacterial agents and anti-cancer chemotherapeutic agents
Extensive evidence supports the hypothesis that topoisomerase poisons kill cells because of the DNA damage generated by the covalent complex rather than by depriving cells of an essential enzymatic activity [3, 6]
Eukaryotic topoisomerase I is the target of camptothecins, whereas eukaryotic topoisomerase II is the target of numerous agents including DNA intercalating agents such as the anthracycline doxorubicin and nonintercalating agents such as the epipodophyllotoxin etoposide

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Plasmids—All experiments for assessing drug sensitivity were carried out in JN362a (MATa ura leu trp his ade ISE2) or its isogenic rad52Ϫ and top derivatives, JN362at, (MATa ura leu trp his ade ISE2 top2-4), JN394 (MATa ura leu trp his ade ISE2 rad52::LEU2), and JN394t2-4 (MATa ura leu trp his ade ISE2 rad52::LEU2 top). For the construction of the T744A mutant, the following pair of oligonucleotides were used: 5Ј-CAG TCA TTG GCA CAA GCT ATT ATT GGG CTA GC-3Ј and 5Ј-GC TAG CCC AAT AAT AGC TTG TGC CAA TGA CTG-3Ј. Measurement of Drug Sensitivity in Yeast Cells—Drug sensitivity in yeast cells was carried out as described previously [26, 32,33,34]. Measurement of Topoisomerase II Binding to DNA—Binding of topoisomerase II to DNA was performed using gel mobility shift assay as described previously with minor modifications [25, 38]. A 10-␮l reaction volume containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM KCl, 2.5% glycerol, 88.5 ng of pUC18, and varying amounts of purified Top protein was incubated at 30 °C for 6 min. All graphics were constructed using Insight II (version 2.97/95.0)

RESULTS

Wild type

Cipro Cipro Norflox Norflox Oxo acid Oxo acid

DISCUSSION