Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length.
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.
Fahrer
Jörg
J
Department of Biology, Molecular Toxicology Group, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany.
Kranaster
Ramon
R
Altmeyer
Matthias
M
Marx
Andreas
A
Bürkle
Alexander
A
D019921:Biotinylation; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D007700:Kinetics; D011064:Poly Adenosine Diphosphate Ribose / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein / Q000378:metabolism
D016159:Tumor Suppressor Protein p53; D051760:Xeroderma Pigmentosum Group A Protein; D011064:Poly Adenosine Diphosphate Ribose