Repair of topoisomerase-mediated DNA damage in bacteriophage T4.
Type II topoisomerase inhibitors are used to treat both tumors and bacterial infections. These inhibitors stabilize covalent DNA-topoisomerase cleavage complexes that ultimately cause lethal DNA damage. A functional recombinational repair apparatus decreases sensitivity to these drugs, suggesting that topoisomerase-mediated DNA damage is amenable to such repair. Using a bacteriophage T4 model system, we have developed a novel in vivo plasmid-based assay that allows physical analysis of the repair products from one particular topoisomerase cleavage site. We show that the antitumor agent 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) stabilizes the T4 type II topoisomerase at the strong topoisomerase cleavage site on the plasmid, thereby stimulating recombinational repair. The resulting m-AMSA-dependent repair products do not form in the absence of functional topoisomerase and appear at lower drug concentrations with a drug-hypersensitive topoisomerase mutant. The appearance of repair products requires that the plasmid contain a T4 origin of replication. Finally, genetic analyses demonstrate that repair product formation is absolutely dependent on genes 32 and 46, largely dependent on genes uvsX and uvsY, and only partly dependent on gene 49. Very similar genetic requirements are observed for repair of endonuclease-generated double-strand breaks, suggesting mechanistic similarity between the two repair pathways.
Stohr
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BA
Departments of Microbiology and Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Kreuzer
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KN
D000677:Amsacrine / Q000494:pharmacology; D017122:Bacteriophage T4 / Q000235:genetics*; D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004279:DNA, Viral / Q000187:drug effects / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D011995:Recombination, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D000677:Amsacrine; D004250:DNA Topoisomerases, Type II