Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I.
Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding.
Colley
William C
WC
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
van der Merwe
Marie
M
Vance
John R
JR
Burgin
Alex B
AB
Bjornsti
Mary-Ann
MA
D001216:Asparagine; D001665:Binding Sites; D002166:Camptothecin / Q000378:metabolism / Q000494:pharmacology; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine; D016296:Mutagenesis; D010649:Phenylalanine; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship; D014443:Tyrosine
D004278:DNA, Superhelical; D004791:Enzyme Inhibitors; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; D001216:Asparagine; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin