Substitution of Conserved Residues within the Active Site Alters the Cleavage Religation Equilibrium of DNA Topoisomerase I

Abstract

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 Top1N726Hp⅐DNA 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.DNA topoisomerases catalyze changes in the linkage of DNA strands, playing critical roles in DNA replication, transcription, and recombination, as well as chromosome condensation and segregation (reviewed in Refs. 1-3). Alterations in DNA topology are catalyzed in reactions characterized by the formation of a covalent enzyme-DNA intermediate. Eukaryotic DNA topoisomerase I (Top1p) 1 is a type IB enzyme that transiently cleaves a single strand of duplex DNA, while forming a tyrosyl linkage with the 3Ј-phosphoryl end of the cleaved DNA (1-3). Rotation of the non-covalently held DNA around the nonscissile DNA strand allows for the relaxation of positive or negative supercoils. In a second transesterification reaction, the 5Ј-OH nucleophile attacks the phosphotyrosyl linkage to religate the DNA.In eukaryotes, the nuclear type IB enzyme, encoded by the TOP1 gene, is highly conserved in terms of amino acid sequence and catalytic mechanism (1-5). The nuclear enzyme is also the cellular target of several antitumor agents, such as camptothecin (CPT) and indolocarbazole analogs (reviewed in Refs. 6 -9). The CPT analogs, Topotecan and SN-38 (the active metabolite of CPT-11), have shown remarkable antitumor activity against a broad range of pediatric and adult malignancies (10). These drugs target Top1p by reversibly ...

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

“…In addition, the accumulation of enzyme-DNA intermediates can also result from mutation-induced alterations in DNA cleavage/religation; e.g. the self-poisoning Top1 mutants Top1T722A and Top1N726H (46,47). The best example of a naturally occurring mutation, the Tdp1H493R mutant identified in human SCAN1 patients, is associated with a progressive cerebellar atrophy that presents in the second decade of life (13).…”

“…Thus, the increased flexibility or spatial distortion of the Tdp1 active site, which impairs the resolution of the reaction intermediates, may also trigger alternative Rad9-dependent DNA repair pathways to induce even more toxic DNA lesions. These results are reminiscent of CPT poisoning of Top1-DNA complexes or of the self-poisoning mutants Top1N722H and Top1T718A, which accumulate Top1-DNA complexes via distinct defects in catalysis (46,47). Top1N722H exhibits an enhanced rate of DNA cleavage (formation of the covalent Top1-DNA complex), and Top1T718A displays a reduced religation rate (slower resolution of the covalent intermediate), whereas the double mutant is even more toxic than either single mutant enzyme.…”

Enzyme Architecture and Flexibility Affect DNA Topoisomerase I Function

“…DNA topoisomerase I activity was assayed in plasmid DNA relaxation reaction as described (99,103). Briefly, serial 10-fold dilutions of Top1 proteins, corrected for concentration, were incubated at 30 ˚C for 30 minutes with 0.3 µg of negatively supercoiled plasmid pHC624 DNA in 20 mM Tris (pH 7.5), 10 mM MgCl 2 , 0.1 mM EDTA, 50 µg/ml gelatin and KCl concentrations ranging from 50 to 200 mM.…”

“…Wild-type and mutant enzyme sensitivity to CPT was determined in DNA cleavage assays as described (99,103 …”

“…Relative rates of Top1-catalyzed DNA religation were assessed using two oligonucleotide-based religation assays adapted from Colley et al, (103). In the postannealed religation substrate reactions, the same 5'-end labeled DNA suicide substrate described above, which contains the DNA scissile strand (5'-GATCTAAAAGACTT↓GG-3'), was incubated with equal concentrations of Top1 proteins in Cleavage buffer at room temperature for 15 minutes to generate Top1-DNA cleavage complexes.…”