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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

An altered CPT sensitivity was shown by T729 mutants when expressed in yeast. Exponential cultures of EKY3 (top1Δ) cells transformed with vector control YCp50, YCpGAL1-hTOP1, YCpGAL1-htop1Ala653Pro,Thr718Ala [a known CPT resistant mutant (29)], YCpGAL1-htop1Thr729Ala, YCpGAL1-htop1Thr729Pro, YCpGAL1-htop1Thr729Lys and YCpGAL1-htop1Thr729Glu were serially 10-fold diluted and spotted onto SC-uracil plates supplemented with dextrose (Dex) or galactose (Gal) and, as indicated, CPT. Cell viability was assessed following incubation at 30°C.
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Figure 1: An altered CPT sensitivity was shown by T729 mutants when expressed in yeast. Exponential cultures of EKY3 (top1Δ) cells transformed with vector control YCp50, YCpGAL1-hTOP1, YCpGAL1-htop1Ala653Pro,Thr718Ala [a known CPT resistant mutant (29)], YCpGAL1-htop1Thr729Ala, YCpGAL1-htop1Thr729Pro, YCpGAL1-htop1Thr729Lys and YCpGAL1-htop1Thr729Glu were serially 10-fold diluted and spotted onto SC-uracil plates supplemented with dextrose (Dex) or galactose (Gal) and, as indicated, CPT. Cell viability was assessed following incubation at 30°C.

Mentions: To assess the in vivo consequences of htop1Thr729 mutant expression, the viability and CPT sensitivity of top1Δ; cells transformed with GAL1-top1 constructs were assayed (Figure 1). The top1Δ; cells expressing human TOP1 exhibit a severe drop in viability in the presence of CPT, whereas cells expressing Thr729 mutants displayed different level of CPT sensitivity.Figure 1.


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)

An altered CPT sensitivity was shown by T729 mutants when expressed in yeast. Exponential cultures of EKY3 (top1Δ) cells transformed with vector control YCp50, YCpGAL1-hTOP1, YCpGAL1-htop1Ala653Pro,Thr718Ala [a known CPT resistant mutant (29)], YCpGAL1-htop1Thr729Ala, YCpGAL1-htop1Thr729Pro, YCpGAL1-htop1Thr729Lys and YCpGAL1-htop1Thr729Glu were serially 10-fold diluted and spotted onto SC-uracil plates supplemented with dextrose (Dex) or galactose (Gal) and, as indicated, CPT. Cell viability was assessed following incubation at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: An altered CPT sensitivity was shown by T729 mutants when expressed in yeast. Exponential cultures of EKY3 (top1Δ) cells transformed with vector control YCp50, YCpGAL1-hTOP1, YCpGAL1-htop1Ala653Pro,Thr718Ala [a known CPT resistant mutant (29)], YCpGAL1-htop1Thr729Ala, YCpGAL1-htop1Thr729Pro, YCpGAL1-htop1Thr729Lys and YCpGAL1-htop1Thr729Glu were serially 10-fold diluted and spotted onto SC-uracil plates supplemented with dextrose (Dex) or galactose (Gal) and, as indicated, CPT. Cell viability was assessed following incubation at 30°C.
Mentions: To assess the in vivo consequences of htop1Thr729 mutant expression, the viability and CPT sensitivity of top1Δ; cells transformed with GAL1-top1 constructs were assayed (Figure 1). The top1Δ; cells expressing human TOP1 exhibit a severe drop in viability in the presence of CPT, whereas cells expressing Thr729 mutants displayed different level of CPT sensitivity.Figure 1.

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