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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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Thr729 mutants are catalytically active in vitro. A total of 120 pmol of purified hTop1p, hTop1Thr729Ala, hTop1Thr729Pro, hTop1Thr729Lys and hTop1Thr729Glu were serially 10-fold diluted and incubated in DNA relaxation assays with negatively supercoiled plasmid DNA. Following incubation at 37°C for 30 min, the reaction products were resolved in agarose gels and visualized after staining with ethidium bromide. C indicates no enzyme control.
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Figure 2: Thr729 mutants are catalytically active in vitro. A total of 120 pmol of purified hTop1p, hTop1Thr729Ala, hTop1Thr729Pro, hTop1Thr729Lys and hTop1Thr729Glu were serially 10-fold diluted and incubated in DNA relaxation assays with negatively supercoiled plasmid DNA. Following incubation at 37°C for 30 min, the reaction products were resolved in agarose gels and visualized after staining with ethidium bromide. C indicates no enzyme control.

Mentions: To assess whether the imposed substitutions in the 729 position could alter the relaxation activity of the enzyme, the mutants were purified from galactose-induced yeast top1Δ cells, as described in the Materials and methods section and tested for their specific activity. Serial dilutions of equal protein concentrations were incubated with negatively supercoiled plasmid DNA and the reaction products resolved in agarose gels. As illustrated in Figure 2, at optimum salt condition for the wild-type enzyme (i.e. 150 mM), the specific activity of htopIThr729Glu was significantly reduced, while htop1Thr729Ala, htop1Thr729Pro and htop1Thr729Lys show an overall activity quite comparable to that of hTop1p. The observed reduction of htop1Thr729Glu specific activity could result from several alterations in enzyme catalytic cycle, including DNA association, cleavage, strand rotation and ligation. To discriminate between these possibilities, we analyzed step by step the catalytic cycle of the mutants and compared it to that of the wild-type enzyme.Figure 2.


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)

Thr729 mutants are catalytically active in vitro. A total of 120 pmol of purified hTop1p, hTop1Thr729Ala, hTop1Thr729Pro, hTop1Thr729Lys and hTop1Thr729Glu were serially 10-fold diluted and incubated in DNA relaxation assays with negatively supercoiled plasmid DNA. Following incubation at 37°C for 30 min, the reaction products were resolved in agarose gels and visualized after staining with ethidium bromide. C indicates no enzyme control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2553582&req=5

Figure 2: Thr729 mutants are catalytically active in vitro. A total of 120 pmol of purified hTop1p, hTop1Thr729Ala, hTop1Thr729Pro, hTop1Thr729Lys and hTop1Thr729Glu were serially 10-fold diluted and incubated in DNA relaxation assays with negatively supercoiled plasmid DNA. Following incubation at 37°C for 30 min, the reaction products were resolved in agarose gels and visualized after staining with ethidium bromide. C indicates no enzyme control.
Mentions: To assess whether the imposed substitutions in the 729 position could alter the relaxation activity of the enzyme, the mutants were purified from galactose-induced yeast top1Δ cells, as described in the Materials and methods section and tested for their specific activity. Serial dilutions of equal protein concentrations were incubated with negatively supercoiled plasmid DNA and the reaction products resolved in agarose gels. As illustrated in Figure 2, at optimum salt condition for the wild-type enzyme (i.e. 150 mM), the specific activity of htopIThr729Glu was significantly reduced, while htop1Thr729Ala, htop1Thr729Pro and htop1Thr729Lys show an overall activity quite comparable to that of hTop1p. The observed reduction of htop1Thr729Glu specific activity could result from several alterations in enzyme catalytic cycle, including DNA association, cleavage, strand rotation and ligation. To discriminate between these possibilities, we analyzed step by step the catalytic cycle of the mutants and compared it to that of the wild-type enzyme.Figure 2.

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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