Journal article: Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length

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2007
Editors
Fahrer, Jörg
Altmeyer, Matthias
Journal article
Abstract
Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.
540 Chemistry
Published in
Nucleic Acids Research ; 35 (2007), 21. - e143. - ISSN 0305-1048. - eISSN 1362-4962
Cite This
ISO 690FAHRER, Jörg, Ramon KRANASTER, Matthias ALTMEYER, Andreas MARX, Alexander BÜRKLE, 2007. Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length. In: Nucleic Acids Research. 35(21), e143. ISSN 0305-1048. eISSN 1362-4962
BibTex
@article{Fahrer2007Quant-9548,
year={2007},
title={Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length},
number={21},
volume={35},
issn={0305-1048},
journal={Nucleic Acids Research},
author={Fahrer, Jörg and Kranaster, Ramon and Altmeyer, Matthias and Marx, Andreas and Bürkle, Alexander},
note={Article Number: e143}
}

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<dcterms:abstract xml:lang="eng">Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.</dcterms:abstract>
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