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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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The htop1Thr729Glu and htop1Thr729Lys mutant enzymes exhibit a lower salt optimum than wild-type hTop1p in plasmid DNA relaxation assays. (A) Limiting amounts of purified hTop1p, htop1Thr729Ala, htop1Thr729Pro, htop1Thr729Lys or htop1Thr729Glu were serially 10-fold diluted and incubated with plasmid DNA at 37°C, in the presence of the indicated concentration of KCl. The reaction products were resolved in agarose gels as described in Figure 2. (B) The percentage of relaxed plasmid DNA topoisomers obtained with hTop1p (filled square), htop1Thr729Ala (inverted filled triangle), htop1Thr729Pro (filled triangle), htop1Thr729Lys (open circle) and htop1The729Glu (open diamond) at the indicated concentration of KCl were determined in a minimum of three independent assays. Error bars indicate SDs from the mean.
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Figure 3: The htop1Thr729Glu and htop1Thr729Lys mutant enzymes exhibit a lower salt optimum than wild-type hTop1p in plasmid DNA relaxation assays. (A) Limiting amounts of purified hTop1p, htop1Thr729Ala, htop1Thr729Pro, htop1Thr729Lys or htop1Thr729Glu were serially 10-fold diluted and incubated with plasmid DNA at 37°C, in the presence of the indicated concentration of KCl. The reaction products were resolved in agarose gels as described in Figure 2. (B) The percentage of relaxed plasmid DNA topoisomers obtained with hTop1p (filled square), htop1Thr729Ala (inverted filled triangle), htop1Thr729Pro (filled triangle), htop1Thr729Lys (open circle) and htop1The729Glu (open diamond) at the indicated concentration of KCl were determined in a minimum of three independent assays. Error bars indicate SDs from the mean.

Mentions: We first assessed the effects of ionic strength on mutant enzyme catalysis. hTop1p activity exhibits a salt optimum around 150 mM. Deviations from this value have previously been shown to correspond to increased or decreased DNA binding, for salt optimum more or less than 150 mM, respectively. Limiting amounts of hTop1p or htop1 mutants were incubated in plasmid relaxation assays containing increasing concentrations of KCl (Figure 3A). The percentage of relaxed plasmid DNA topoisomers produced ([relaxed DNA topoisomers]/[relaxed + supercoiled DNA topoisomers]) following incubation with the enzymes was quantified and plotted as a function of salt concentration (Figure 3B). Thr729 mutants display significant differences in their requirements for optimum of KCl concentration. In fact, while htopIThr729Ala shows a maximum in the catalytic activity comparable to that of the wild-type enzyme (150 mM KCl), DNA relaxation catalyzed by htop1Thr729Pro and htop1Thr729Lys was enhanced at lower salt concentrations (100 mM KCl). The minimum KCl concentration is shown by the Thr729Glu mutant where the optimum is reached at 25 mM KCl. These results are consistent with a general lower affinity effect of htop1Thr729 mutants for DNA than wild-type enzyme that becomes maximum in the case of the htop1Thr729Glu protein.Figure 3.


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)

The htop1Thr729Glu and htop1Thr729Lys mutant enzymes exhibit a lower salt optimum than wild-type hTop1p in plasmid DNA relaxation assays. (A) Limiting amounts of purified hTop1p, htop1Thr729Ala, htop1Thr729Pro, htop1Thr729Lys or htop1Thr729Glu were serially 10-fold diluted and incubated with plasmid DNA at 37°C, in the presence of the indicated concentration of KCl. The reaction products were resolved in agarose gels as described in Figure 2. (B) The percentage of relaxed plasmid DNA topoisomers obtained with hTop1p (filled square), htop1Thr729Ala (inverted filled triangle), htop1Thr729Pro (filled triangle), htop1Thr729Lys (open circle) and htop1The729Glu (open diamond) at the indicated concentration of KCl were determined in a minimum of three independent assays. Error bars indicate SDs from the mean.
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Figure 3: The htop1Thr729Glu and htop1Thr729Lys mutant enzymes exhibit a lower salt optimum than wild-type hTop1p in plasmid DNA relaxation assays. (A) Limiting amounts of purified hTop1p, htop1Thr729Ala, htop1Thr729Pro, htop1Thr729Lys or htop1Thr729Glu were serially 10-fold diluted and incubated with plasmid DNA at 37°C, in the presence of the indicated concentration of KCl. The reaction products were resolved in agarose gels as described in Figure 2. (B) The percentage of relaxed plasmid DNA topoisomers obtained with hTop1p (filled square), htop1Thr729Ala (inverted filled triangle), htop1Thr729Pro (filled triangle), htop1Thr729Lys (open circle) and htop1The729Glu (open diamond) at the indicated concentration of KCl were determined in a minimum of three independent assays. Error bars indicate SDs from the mean.
Mentions: We first assessed the effects of ionic strength on mutant enzyme catalysis. hTop1p activity exhibits a salt optimum around 150 mM. Deviations from this value have previously been shown to correspond to increased or decreased DNA binding, for salt optimum more or less than 150 mM, respectively. Limiting amounts of hTop1p or htop1 mutants were incubated in plasmid relaxation assays containing increasing concentrations of KCl (Figure 3A). The percentage of relaxed plasmid DNA topoisomers produced ([relaxed DNA topoisomers]/[relaxed + supercoiled DNA topoisomers]) following incubation with the enzymes was quantified and plotted as a function of salt concentration (Figure 3B). Thr729 mutants display significant differences in their requirements for optimum of KCl concentration. In fact, while htopIThr729Ala shows a maximum in the catalytic activity comparable to that of the wild-type enzyme (150 mM KCl), DNA relaxation catalyzed by htop1Thr729Pro and htop1Thr729Lys was enhanced at lower salt concentrations (100 mM KCl). The minimum KCl concentration is shown by the Thr729Glu mutant where the optimum is reached at 25 mM KCl. These results are consistent with a general lower affinity effect of htop1Thr729 mutants for DNA than wild-type enzyme that becomes maximum in the case of the htop1Thr729Glu protein.Figure 3.

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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