Limits...
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.

Show MeSH
Structure modifications induced by the Thr729 mutations. Panel A1: helix 16, in core domain, and helix 21, in the C-terminal domain are highlighted in red and light blue colours, respectively. Panel B1–C1–D1: only the helices are shown in representative snapshots of the htopIThr729Lys, with hTopIp and htopIThr729Pro simulations, respectively. The side chains of Thr606 and Trp732, in helixes 16 and 17, respectively, are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line. Panel A2: helix 17, in core domain, and helices 20 and 21, in the C-terminal domain are highlighted in red and light blue colours, respectively. Panel B2–C2–D2: only the helices are shown in representative snapshots of the htopIThr729Lys, hTopIp and htopIThr729Pro simulations, respectively. The side chains of Lys720, in helix 20, of the mutated residue 729, in helix 21, and of Tyr619, in helix 17 are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Structure modifications induced by the Thr729 mutations. Panel A1: helix 16, in core domain, and helix 21, in the C-terminal domain are highlighted in red and light blue colours, respectively. Panel B1–C1–D1: only the helices are shown in representative snapshots of the htopIThr729Lys, with hTopIp and htopIThr729Pro simulations, respectively. The side chains of Thr606 and Trp732, in helixes 16 and 17, respectively, are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line. Panel A2: helix 17, in core domain, and helices 20 and 21, in the C-terminal domain are highlighted in red and light blue colours, respectively. Panel B2–C2–D2: only the helices are shown in representative snapshots of the htopIThr729Lys, hTopIp and htopIThr729Pro simulations, respectively. The side chains of Lys720, in helix 20, of the mutated residue 729, in helix 21, and of Tyr619, in helix 17 are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line.

Mentions: As shown in Figure 8, panel C2, in the wild-type protein a direct hydrogen bond between helix 21 and helix 17 is formed by the Thr729 and Tyr619. The results obtained by the dynamic simulation described in the accompanying paper have shown that in the case of the Thr729Pro mutant the direct hydrogen bond became lost without altering the relative positions of the two helices (Figure 8, panel D2). On the contrary, the Thr729Lys mutation provokes a more dramatic effect. As it can be seen in Figure 8, panel B2, the loss of the direct hydrogen bond is followed by the moving away of helix 21 from helix 17 with a direct effect on the drug-binding pocket.Figure 8.


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)

Structure modifications induced by the Thr729 mutations. Panel A1: helix 16, in core domain, and helix 21, in the C-terminal domain are highlighted in red and light blue colours, respectively. Panel B1–C1–D1: only the helices are shown in representative snapshots of the htopIThr729Lys, with hTopIp and htopIThr729Pro simulations, respectively. The side chains of Thr606 and Trp732, in helixes 16 and 17, respectively, are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line. Panel A2: helix 17, in core domain, and helices 20 and 21, in the C-terminal domain are highlighted in red and light blue colours, respectively. Panel B2–C2–D2: only the helices are shown in representative snapshots of the htopIThr729Lys, hTopIp and htopIThr729Pro simulations, respectively. The side chains of Lys720, in helix 20, of the mutated residue 729, in helix 21, and of Tyr619, in helix 17 are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Structure modifications induced by the Thr729 mutations. Panel A1: helix 16, in core domain, and helix 21, in the C-terminal domain are highlighted in red and light blue colours, respectively. Panel B1–C1–D1: only the helices are shown in representative snapshots of the htopIThr729Lys, with hTopIp and htopIThr729Pro simulations, respectively. The side chains of Thr606 and Trp732, in helixes 16 and 17, respectively, are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line. Panel A2: helix 17, in core domain, and helices 20 and 21, in the C-terminal domain are highlighted in red and light blue colours, respectively. Panel B2–C2–D2: only the helices are shown in representative snapshots of the htopIThr729Lys, hTopIp and htopIThr729Pro simulations, respectively. The side chains of Lys720, in helix 20, of the mutated residue 729, in helix 21, and of Tyr619, in helix 17 are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line.
Mentions: As shown in Figure 8, panel C2, in the wild-type protein a direct hydrogen bond between helix 21 and helix 17 is formed by the Thr729 and Tyr619. The results obtained by the dynamic simulation described in the accompanying paper have shown that in the case of the Thr729Pro mutant the direct hydrogen bond became lost without altering the relative positions of the two helices (Figure 8, panel D2). On the contrary, the Thr729Lys mutation provokes a more dramatic effect. As it can be seen in Figure 8, panel B2, the loss of the direct hydrogen bond is followed by the moving away of helix 21 from helix 17 with a direct effect on the drug-binding pocket.Figure 8.

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