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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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Cleavage–religation equilibrium. Equal concentrations of hTop1p, htop1Thr729Al, htop1Thr729Pro, htop1Thr729Lys or htop1Thr729Glu were incubated with a single 32P-end-labeled DNA fragment in absence or presence of CPT as described in Materials and methods section. After 30 min the reactions were stopped with 0.5% SDS and the cleavage products resolved by urea/PAGE. C indicates no enzyme control. The arrow indicates the position of a high affinity cleavage site.
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Figure 4: Cleavage–religation equilibrium. Equal concentrations of hTop1p, htop1Thr729Al, htop1Thr729Pro, htop1Thr729Lys or htop1Thr729Glu were incubated with a single 32P-end-labeled DNA fragment in absence or presence of CPT as described in Materials and methods section. After 30 min the reactions were stopped with 0.5% SDS and the cleavage products resolved by urea/PAGE. C indicates no enzyme control. The arrow indicates the position of a high affinity cleavage site.

Mentions: Equal concentrations of hTop1p and mutants proteins were incubated with a 900-bp 3′-end-labeled DNA fragment. Following incubation at 37°C for 30 min, the cleavable complexes were trapped by SDS and the cleaved DNA fragments were resolved in a denaturing polyacrylamide gel, Figure 4. Under these conditions, the relative intensity and distribution of cleaved DNA fragments can be used to assess mutation and/or CPT-induced alterations in the steady-state levels of covalent enzyme–DNA intermediates.Figure 4.


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)

Cleavage–religation equilibrium. Equal concentrations of hTop1p, htop1Thr729Al, htop1Thr729Pro, htop1Thr729Lys or htop1Thr729Glu were incubated with a single 32P-end-labeled DNA fragment in absence or presence of CPT as described in Materials and methods section. After 30 min the reactions were stopped with 0.5% SDS and the cleavage products resolved by urea/PAGE. C indicates no enzyme control. The arrow indicates the position of a high affinity cleavage site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553582&req=5

Figure 4: Cleavage–religation equilibrium. Equal concentrations of hTop1p, htop1Thr729Al, htop1Thr729Pro, htop1Thr729Lys or htop1Thr729Glu were incubated with a single 32P-end-labeled DNA fragment in absence or presence of CPT as described in Materials and methods section. After 30 min the reactions were stopped with 0.5% SDS and the cleavage products resolved by urea/PAGE. C indicates no enzyme control. The arrow indicates the position of a high affinity cleavage site.
Mentions: Equal concentrations of hTop1p and mutants proteins were incubated with a 900-bp 3′-end-labeled DNA fragment. Following incubation at 37°C for 30 min, the cleavable complexes were trapped by SDS and the cleaved DNA fragments were resolved in a denaturing polyacrylamide gel, Figure 4. Under these conditions, the relative intensity and distribution of cleaved DNA fragments can be used to assess mutation and/or CPT-induced alterations in the steady-state levels of covalent enzyme–DNA intermediates.Figure 4.

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
Related in: MedlinePlus