Nucleic Acids Research Advance Access originally published online on November 8, 2007
Nucleic Acids Research 2007 35(21):e143; doi:10.1093/nar/gkm944
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Nucleic Acids Research, 2007, Vol. 35, No. 21 e143
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length
1Department of Biology, Molecular Toxicology Group and 2Department of Chemistry, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany
*To whom correspondence should be addressed. Tel: +49 7531 884035; Fax: +49 7531 884033; Email: alexander.buerkle{at}uni-konstanz.de
Received August 23, 2007. Revised October 9, 2007. Accepted October 12, 2007.
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.
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