Abstract
BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear.MethodsThe three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation.ResultsThe three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine.ConclusionsThese results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.
Highlights
DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands
Through a combined experimental and simulative approach, we provide evidence that the CPT resistance of the mutant is due to a fast religation rate coupled to a loss of correlation between the linker and the C-terminal domain confirming the crucial role of the linker domain in controlling Human topoisomerase IB (hTop1) drug sensitivity
Glu710Gly mutant is resistant to CPT in vivo and in vitro The mutations (Leu617Ile, Arg621His and Glu710Gly) found in the CPT-11 resistant HCT116 clones by Gongora et al [24] have been introduced in the single copy yeast plasmid (YCp) expressing hTopI under the GAL1 promoter
Summary
DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, inducing cell death. Separation of the two strands of the double helix generates tensions and other structure of the N-terminal truncated protein (Topo70), together with proteolytic experiments, has shown that the enzyme is composed of four different domains: the N-terminal domain (residues 1–214), the core domain (215–635), the linker domain (636–712), and the C-terminal domain (713–765) [3,4,5]. HTop catalyzes DNA relaxation by transiently cleaving, passing, and religating one strand of the DNA double helix. After changing the linking number, a second nucleophilic attack, driven by the 5′-hydroxy DNA end, restores an intact double-stranded DNA, and the enzyme is released [5]
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