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A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance.

Losasso C, Cretaio E, Fiorani P, D'Annessa I, Chillemi G, Benedetti P - Nucleic Acids Res. (2008)

Bottom Line: The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA.To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro).We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Padova, Via U. Bassi 58/B, Padua 35131, Italy.

ABSTRACT
Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.

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Time course of plasmid DNA relaxation catalyzed by hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu. Equal concentrations of hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu were incubated with supercoiled plasmid DNA in the presence of 150 mM KCl in the presence of an excess of DNA (A) or protein (B). At the times indicated, aliquots were treated with 0.5% SDS and the reaction products were resolved in agarose gels as in Figure 2.
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Figure 6: Time course of plasmid DNA relaxation catalyzed by hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu. Equal concentrations of hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu were incubated with supercoiled plasmid DNA in the presence of 150 mM KCl in the presence of an excess of DNA (A) or protein (B). At the times indicated, aliquots were treated with 0.5% SDS and the reaction products were resolved in agarose gels as in Figure 2.

Mentions: The failure in the recognition of a suicide DNA substrate, the absence of any trapped DNA cleaved products in the presence of CPT and the reduced capability to relax the DNA in physiologic ionic strength conditions by the htop1Thr729Glu enzyme, are strictly related to one of the steps involved in the catalytic cycle of the enzyme: the equilibrium DNA binding/DNA dissociation. To address this point, we analyzed the kinetics of DNA relaxation of the three CPT-resistant mutants htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu, compared to that of the wild-type enzyme. The experiments were conducted in the presence of 150 mM KCl, using equal protein amounts with an excess or a deficit of negatively supercoiled plasmid DNA compared to the enzyme (DNA/enzyme ratio 3 : 1 or 1 : 3) (Figure 6). In the presence of excess DNA, all the enzyme molecules are assumed to interact with the substrate. In these conditions, the speed of the relaxation activity can be expressed as a function of the enzyme Km. As shown in Figure 6, both the htop1Thr729Lys and htop1Thr729Glu enzymes relax the DNA more slowly than wild-type accomplishing the same final relaxation level.Figure 6.


A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance.

Losasso C, Cretaio E, Fiorani P, D'Annessa I, Chillemi G, Benedetti P - Nucleic Acids Res. (2008)

Time course of plasmid DNA relaxation catalyzed by hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu. Equal concentrations of hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu were incubated with supercoiled plasmid DNA in the presence of 150 mM KCl in the presence of an excess of DNA (A) or protein (B). At the times indicated, aliquots were treated with 0.5% SDS and the reaction products were resolved in agarose gels as in Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553582&req=5

Figure 6: Time course of plasmid DNA relaxation catalyzed by hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu. Equal concentrations of hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu were incubated with supercoiled plasmid DNA in the presence of 150 mM KCl in the presence of an excess of DNA (A) or protein (B). At the times indicated, aliquots were treated with 0.5% SDS and the reaction products were resolved in agarose gels as in Figure 2.
Mentions: The failure in the recognition of a suicide DNA substrate, the absence of any trapped DNA cleaved products in the presence of CPT and the reduced capability to relax the DNA in physiologic ionic strength conditions by the htop1Thr729Glu enzyme, are strictly related to one of the steps involved in the catalytic cycle of the enzyme: the equilibrium DNA binding/DNA dissociation. To address this point, we analyzed the kinetics of DNA relaxation of the three CPT-resistant mutants htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu, compared to that of the wild-type enzyme. The experiments were conducted in the presence of 150 mM KCl, using equal protein amounts with an excess or a deficit of negatively supercoiled plasmid DNA compared to the enzyme (DNA/enzyme ratio 3 : 1 or 1 : 3) (Figure 6). In the presence of excess DNA, all the enzyme molecules are assumed to interact with the substrate. In these conditions, the speed of the relaxation activity can be expressed as a function of the enzyme Km. As shown in Figure 6, both the htop1Thr729Lys and htop1Thr729Glu enzymes relax the DNA more slowly than wild-type accomplishing the same final relaxation level.Figure 6.

Bottom Line: The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA.To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro).We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Padova, Via U. Bassi 58/B, Padua 35131, Italy.

ABSTRACT
Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.

Show MeSH