title: Mutations of the bacteriophage T4 type II DNA topoisomerase that alter sensitivity to antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide and an antibacterial quinolone.
abstract: Various antitumor and antibacterial agents target type II DNA topoisomerases, stabilizing a cleaved DNA reaction intermediate and thereby converting topoisomerase into a cellular poison. Two 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)-resistant bacteriophage T4 topoisomerases have previously been characterized biochemically, and we have now determined the sequence of the causative mutations. In one case, a mutation (E457K) in a conserved domain of gp39 (ATPase subunit) causes resistance to antitumor agent m-AMSA but hypersensitivity to the quinolone oxolinic acid. In the second case, a combination of two amino acid substitutions (S79F and G269V) in gp52 (DNA-cleaving subunit) causes resistance to both m-AMSA and oxolinic acid. The S79F mutation is responsible for drug resistance, whereas the G269V mutation suppresses a topoisomerase deficiency caused by S79F. Surprisingly, the G269V mutation by itself causes a dramatic hypersensitivity to both inhibitors, defining a new class of topoisomerase mutants. Because S79 and the adjacent N78 are homologous to two key residues of DNA gyrase that affect quinolone sensitivity, we generated additional amino acid substitutions at these two positions. The substitutions alter sensitivity to m-AMSA and to oxolinic acid, sometimes in opposite directions. Furthermore, the quinolone sensitivities of the various mutants paralleled those of corresponding gyrase mutants. These results support models in which both quinolones and antitumor agents bind to a conserved site that overlaps the active site of the enzyme.
authors.affiliation: Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: C H
authors.initials: CH
authors.lastname: Freudenreich
authors.forename: C
authors.initials: C
authors.lastname: Chang
authors.forename: K N
authors.initials: KN
authors.lastname: Kreuzer
mesh_terms: D000595:Amino Acid Sequence; D000677:Amsacrine / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004352:Drug Resistance, Microbial; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D010093:Oxolinic Acid / Q000494:pharmacology*; D017354:Point Mutation; D013329:Structure-Activity Relationship; D059005:Topoisomerase II Inhibitors
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; D004250:DNA Topoisomerases, Type II; D010093:Oxolinic Acid