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

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

doi:10.1186/s13046-014-0071-z

Abstract

BackgroundDNA 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.MethodsUsing site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures.ResultsMutations 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.ConclusionsIt 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.

Highlights

DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands
Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes, of which PARP-1 is the best characterized member [1]
* Correspondence: graziani@uniroma2.it; paola.fiorani@uniroma2.it; desideri@uniroma2.it †Equal contributors 2Department of Systems Medicine, University of Rome Tor ‘Vergata Via’ Montpellier 1, Rome 00133, Italy 4Institute of Translational Pharmacology, National Research Council, CNR, Via del Fosso del Cavaliere 100, Rome 00133, Italy 1Department of Biology and Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica, Rome 00133, Italy Full list of author information is available at the end of the article (PARs) that are covalently linked to glutamic, aspartic and lysine amino acids of target proteins, such as histones, transcription factors and PARP-1 itself, which is the main acceptor of PARs

Summary

Introduction

DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. PARylated PARP-1 or free PARs, that are highly flexible polymers [4], can non-covalently interact with a variety of proteins containing a PAR binding motif (PBM), which includes basic and hydrophobic residues downstream of a positively charged cluster rich in lysine and arginine [5,6,7,8]. This motif has been found in proteins involved in DNA damage response, chromatin structure, replication and transcription and can be present in different domains of the same target protein

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Conclusion