Mutations of human DNA topoisomerase I at poly (ADP-ribose) binding sites: Modulation of camptothecin activity by ADP-ribose polymers

Cinzia Tesauro, Grazia Graziani, Barbara Arnò, Laura Zuccaro, Alessia Muzi, Ilda D'Annessa, Elettra Santori, Lucio Tentori, Carlo Leonetti, Paola Fiorani, Alessandro Desideri

Research output: Contribution to journalArticle

Abstract

Background: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects. Methods: Using site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly (ADP-ribose) through enzymatic activity and binding procedures. Results: Mutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity. Conclusions: It can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.

Original languageEnglish
Article number71
JournalJournal of Experimental and Clinical Cancer Research
Volume33
Issue number1
DOIs
Publication statusPublished - 2014

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Adenosine Diphosphate Ribose
Poly Adenosine Diphosphate Ribose
Camptothecin
Type I DNA Topoisomerase
Polymers
Binding Sites
Mutation
Poisons
Enzymes
DNA Topoisomerases
DNA
Site-Directed Mutagenesis
Adenosine Diphosphate
Carrier Proteins
human TOP1 protein
Pharmaceutical Preparations
Proteins

Keywords

  • Camptothecin
  • Cleavage
  • PARP inhibitors
  • PARylation
  • Religation rate
  • Topoisomerase I

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Mutations of human DNA topoisomerase I at poly (ADP-ribose) binding sites : Modulation of camptothecin activity by ADP-ribose polymers. / Tesauro, Cinzia; Graziani, Grazia; Arnò, Barbara; Zuccaro, Laura; Muzi, Alessia; D'Annessa, Ilda; Santori, Elettra; Tentori, Lucio; Leonetti, Carlo; Fiorani, Paola; Desideri, Alessandro.

In: Journal of Experimental and Clinical Cancer Research, Vol. 33, No. 1, 71, 2014.

Research output: Contribution to journalArticle

Tesauro, Cinzia ; Graziani, Grazia ; Arnò, Barbara ; Zuccaro, Laura ; Muzi, Alessia ; D'Annessa, Ilda ; Santori, Elettra ; Tentori, Lucio ; Leonetti, Carlo ; Fiorani, Paola ; Desideri, Alessandro. / Mutations of human DNA topoisomerase I at poly (ADP-ribose) binding sites : Modulation of camptothecin activity by ADP-ribose polymers. In: Journal of Experimental and Clinical Cancer Research. 2014 ; Vol. 33, No. 1.
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AU - Tesauro, Cinzia

AU - Graziani, Grazia

AU - Arnò, Barbara

AU - Zuccaro, Laura

AU - Muzi, Alessia

AU - D'Annessa, Ilda

AU - Santori, Elettra

AU - Tentori, Lucio

AU - Leonetti, Carlo

AU - Fiorani, Paola

AU - Desideri, Alessandro

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N2 - Background: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects. Methods: Using site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly (ADP-ribose) through enzymatic activity and binding procedures. Results: Mutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity. Conclusions: It can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.

AB - Background: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects. Methods: Using site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly (ADP-ribose) through enzymatic activity and binding procedures. Results: Mutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity. Conclusions: It can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.

KW - Camptothecin

KW - Cleavage

KW - PARP inhibitors

KW - PARylation

KW - Religation rate

KW - Topoisomerase I

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