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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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Related in: MedlinePlus

Analysis of the religation rate using a suicide substrate. (A) In the oligonucleotide-based suicide substrate, cleavage of the 5′-radiolabeled scissile strand (14 nt) liberates an AG dinucleotide to trap the covalent DNA–hTop1p complex. (B) The percentage of DNA relegation, in the presence and in the absence of CPT, was determined by ImageQuant software and normalized to the total amount of radioactivity in each lane: hTop1p (filled square), htop1Thr729Pro (filled traingle), htop1Thr729Lys.
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Figure 5: Analysis of the religation rate using a suicide substrate. (A) In the oligonucleotide-based suicide substrate, cleavage of the 5′-radiolabeled scissile strand (14 nt) liberates an AG dinucleotide to trap the covalent DNA–hTop1p complex. (B) The percentage of DNA relegation, in the presence and in the absence of CPT, was determined by ImageQuant software and normalized to the total amount of radioactivity in each lane: hTop1p (filled square), htop1Thr729Pro (filled traingle), htop1Thr729Lys.

Mentions: A common strategy to uncouple DNA cleavage from religation is to use an oligonucleotide-based substrate that contains a truncated scissile strand. For example, as depicted in Figure 5A, hTop1p cleavage of a high affinity site within a suicide DNA substrate liberates a dinucleotide and traps the covalent hTop1p–DNA complexes. In the presence of hTop1p, religate DNA molecules accumulate very rapidly in the absence of CPT, whereas the rate of DNA religation is dramatically reduced in the presence of CPT (Figure 5B). In contrast, the rate of DNA religation was substantially enhanced in the case of the htop1Thr729Pro or htop1Thr729Lys. We also tested the htop1Thr729Glu kinetic of religation using the described suicide DNA substrate and other substrates that differ for length and nucleotide sequence. We were unable to detect any cleavage of suicide DNA substrates in the absence and in the presence of an annealed religation strand. These data are consistent with a decrease in mutant enzyme binding to DNA.Figure 5.


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)

Analysis of the religation rate using a suicide substrate. (A) In the oligonucleotide-based suicide substrate, cleavage of the 5′-radiolabeled scissile strand (14 nt) liberates an AG dinucleotide to trap the covalent DNA–hTop1p complex. (B) The percentage of DNA relegation, in the presence and in the absence of CPT, was determined by ImageQuant software and normalized to the total amount of radioactivity in each lane: hTop1p (filled square), htop1Thr729Pro (filled traingle), htop1Thr729Lys.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Analysis of the religation rate using a suicide substrate. (A) In the oligonucleotide-based suicide substrate, cleavage of the 5′-radiolabeled scissile strand (14 nt) liberates an AG dinucleotide to trap the covalent DNA–hTop1p complex. (B) The percentage of DNA relegation, in the presence and in the absence of CPT, was determined by ImageQuant software and normalized to the total amount of radioactivity in each lane: hTop1p (filled square), htop1Thr729Pro (filled traingle), htop1Thr729Lys.
Mentions: A common strategy to uncouple DNA cleavage from religation is to use an oligonucleotide-based substrate that contains a truncated scissile strand. For example, as depicted in Figure 5A, hTop1p cleavage of a high affinity site within a suicide DNA substrate liberates a dinucleotide and traps the covalent hTop1p–DNA complexes. In the presence of hTop1p, religate DNA molecules accumulate very rapidly in the absence of CPT, whereas the rate of DNA religation is dramatically reduced in the presence of CPT (Figure 5B). In contrast, the rate of DNA religation was substantially enhanced in the case of the htop1Thr729Pro or htop1Thr729Lys. We also tested the htop1Thr729Glu kinetic of religation using the described suicide DNA substrate and other substrates that differ for length and nucleotide sequence. We were unable to detect any cleavage of suicide DNA substrates in the absence and in the presence of an annealed religation strand. These data are consistent with a decrease in mutant enzyme binding to DNA.Figure 5.

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