Bacteriophage T4 mutants hypersensitive to an antitumor agent that induces topoisomerase-DNA cleavage complexes.
Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), alpha-gt (alpha-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (beta subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage.
Woodworth
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Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kreuzer
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KN
D000595:Amino Acid Sequence; D000677:Amsacrine / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017122:Bacteriophage T4 / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004251:DNA Transposable Elements; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016914:Ribonuclease H / Q000235:genetics; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000235:genetics
D000970:Antineoplastic Agents; D004251:DNA Transposable Elements; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; D000677:Amsacrine; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D016914:Ribonuclease H; D004250:DNA Topoisomerases, Type II