Mutation of Gly721 alters DNA topoisomerase I active site architecture and sensitivity to camptothecin.
DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anti-cancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended alpha-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short alpha-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this alpha-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Marié
M
van der Merwe
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105. Electronic address: mary-ann.bjornsti@stjude.org.
Mary-Ann
MA
Bjornsti
D001483:Base Sequence; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004264:DNA Topoisomerases, Type I; D005998:Glycine; D002166:Camptothecin