Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin

Abstract

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 anticancer 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 ␣-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 ␣-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this ␣-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 CPTresistant. 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.Eukaryotic DNA topoisomerase 1 (Top1p) 2 alters DNA topology by introducing a transient break in a single strand of the DNA duplex, thereby allowing strand rotation at the site of DNA scission to relieve superhelical tension (1-3). The enzyme plays critical roles in processes such as replication, transcription, recombination, and chromosomal condensation, with a reaction mechanism highly conserved from yeast to human. The active site tyrosine (Tyr 727 in yeast) acts as a nucleophile to cleave a single DNA strand, forming a covalent phosphotyrosyl linkage between the enzyme and the 3Ј-end of the DNA. The free 5Ј-OH end of the scissile strand then rotates about the uncleaved DNA strand in a manner dictated by torsional strain in the DNA. In a second transesterification reaction, the nucleophilic attack by the 5Ј-OH resolves the covalent Top1-DNA intermediate, and the enzyme is liberated from the religated DNA.DNA cleavage by Top1p is, at least in part, rate-limiting, thus ensuring low steady state-levels of the covalent enzyme-DNA complex (4). However, increased concentrations of covalent complexes, either as a consequence of drug action, Top1p mutation, or the formation of DNA adducts, converts Top1p into a cellular poison (5-16). The camptothecin (CPT) class of chemotherapeutics reversibly stabilizes the covalent intermediat...

Analysis of the Active-Site Mechanism of Tyrosyl-DNA Phosphodiesterase I: A Member of the Phospholipase D Superfamily

“…These results demonstrate that hTdp1 functions in yeast in the presence of hTop1, and that the toxic phenotypes of the two yTdp1 mutants H432R and H432N mutants are also manifested by the equivalent hTdp1 mutants. Comparing yeast Top1-Tdp1 to human Top1-Tdp1, it can be seen that the mutant phenotypes are not strictly equivalent, particularly in the case of the His to Asn substitution, but this may be related to species specific differences in hTop1 function in yeast 30; 31; 32; 33 . It has been shown that hTop1 expression in yeast provides the appropriate DNA topoisomerase I activity and conversely, expression of yTop1 in mammalian cells provides the appropriate activity and CPT sensitivity 30; 34 .…”

DNA Topoisomerase I Domain Interactions Impact Enzyme Activity and Sensitivity to Camptothecin

“…In contrast, a Top1D677G/V703I mutant has reduced linker flexibility and is hypersensitive to CPT (29). In addition, replacing Gly 721 with residues estimated to have a higher propensity for ␣ helix formation (Ala or Asp) induced alterations in yeast Top1 active site architecture that increased enzyme sensitivity to CPT (28). In co-crystal structures, the extension of a short ␣ helix within the active site of Topo70 to include Gly 717 coincides with drug binding and a restriction of linker orientation (7).…”

“…Lysates were normalized for total protein, and Top1 protein levels were verified by immunoblotting and chemiluminescence using the M2-FLAG antibody (Sigma). Wild-type and chimeric Top1 were partially purified by phosphocellulose chromatography or purified to homogeneity by phosphocellulose and FLAG affinity chromatography, as described (12,28). Top1 protein fractions were adjusted to a final concentration of 30% glycerol and stored at Ϫ20°C.…”

“…Top1 protein fractions were adjusted to a final concentration of 30% glycerol and stored at Ϫ20°C. Protein integrity was assayed by immunoblot analysis (28,41).…”

Disulfide Cross-links Reveal Conserved Features of DNA Topoisomerase I Architecture and a Role for the N Terminus in Clamp Closure

“…Linker flexibility also impacts mutation-induced alterations in enzymecatalyzed DNA cleavage/religation (24). Indeed, our recent studies suggest that a conserved Gly within the catalytic pocket of Top1 provides a flexible hinge to facilitate linker mobility (26).…”