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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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DNA-binding assay. Increasing amounts of hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu were incubated with an end-labeled 100-nt double-strand DNA fragment as described in Materials and methods section. The percentage of DNA binding as a function of enzyme concentrated is illustrated.
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Figure 7: DNA-binding assay. Increasing amounts of hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu were incubated with an end-labeled 100-nt double-strand DNA fragment as described in Materials and methods section. The percentage of DNA binding as a function of enzyme concentrated is illustrated.

Mentions: In order to quantify the difference in DNA binding by the three obtained CPT-resistant enzymes, a filter binding assay was performed. Increasing amounts of wild-type enzyme and mutants were incubated with a 100-bp labeled DNA fragment, Figure 7A, containing the specific hTop1p recognition sequence as described in the Material and methods section. The reaction mixtures were applied to a nitrocellulose filter and the amount of DNA retained was counted. Figure 7B shows the percent of bound DNA as a function of the enzyme concentration. As it can be appreciated, the amount of DNA bound by the three mutant proteins was significantly reduced relative to the wild-type. Coherently with the reduced catalytic activity observed in the relaxation assays and in the DNA cleavage assay, the DNA binding profile obtained with the single mutant htop1Thr729Lys and htop1Thr729Glu proves that changes in the hydrophobic properties of the C-terminal region of the enzyme radically affect the DNA binding/DNA dissociation equilibrium by hTop1p.Figure 7.


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)

DNA-binding assay. Increasing amounts of hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu were incubated with an end-labeled 100-nt double-strand DNA fragment as described in Materials and methods section. The percentage of DNA binding as a function of enzyme concentrated is illustrated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553582&req=5

Figure 7: DNA-binding assay. Increasing amounts of hTop1p, htop1Thr729Pro, htop1Thr729Lys and htop1Thr729Glu were incubated with an end-labeled 100-nt double-strand DNA fragment as described in Materials and methods section. The percentage of DNA binding as a function of enzyme concentrated is illustrated.
Mentions: In order to quantify the difference in DNA binding by the three obtained CPT-resistant enzymes, a filter binding assay was performed. Increasing amounts of wild-type enzyme and mutants were incubated with a 100-bp labeled DNA fragment, Figure 7A, containing the specific hTop1p recognition sequence as described in the Material and methods section. The reaction mixtures were applied to a nitrocellulose filter and the amount of DNA retained was counted. Figure 7B shows the percent of bound DNA as a function of the enzyme concentration. As it can be appreciated, the amount of DNA bound by the three mutant proteins was significantly reduced relative to the wild-type. Coherently with the reduced catalytic activity observed in the relaxation assays and in the DNA cleavage assay, the DNA binding profile obtained with the single mutant htop1Thr729Lys and htop1Thr729Glu proves that changes in the hydrophobic properties of the C-terminal region of the enzyme radically affect the DNA binding/DNA dissociation equilibrium by hTop1p.Figure 7.

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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