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

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.

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

“…4a), thus demonstrating that the PbR motif is responsible for PAR interaction with Chk1. It is known that the non-covalent binding of proteins to PAR can influence their functionality 52 . We then investigated the impact of PAR binding to Chk1 on the protein's kinase activity.…”

“…The iPOND assay was performed essentially as described 47 Synthesis and fractionation of PAR. PAR was synthesized, purified and HPLC fractionated as described 52,67,68 . Briefly, a reaction mixture containing 150 nM recombinant PARP1 was incubated in 100 mM Tris-HCl pH 7.8, 10 mM MgCl 2 , 10 mM DTT, 60 mg ml À 1 histone H1, 60 mg ml À 1 histone type IIa, 1 mM NAD þ and 50 mg ml À 1 double-stranded activator oligonucleotide 5 0 -GGAATTCC-3 0 for 15 min at 37°C.…”

The oligonucleotide/oligosaccharide-binding fold motif is a poly(ADP-ribose)-binding domain that mediates DNA damage response

“…The mixture was incubated at 30°C for 60 min and stopped by addition of 20 mL ice-cold 20% (wt/vol) trichloroacetic acid. Oligo DNA was removed by DNase I and proteins were digested by proteinase K. Purified PAR was fractionated according to chain length by anion exchange HPLC protocol as described previously (50,51). ADPR chains between 10-and 25-mer were collected as pooled fractions and concentrated for ITC assay.…”

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

“…Generation and purification of PAR and iso-ADP-ribose PAR (or biotin-PAR) was synthesized and purified in vitro according to the previous work as described (Fahrer et al 2007) with some modifications. Briefly, PAR was synthesized in a 15-mL incubation mixture comprising 100 mM Tris-HCl (pH 7.8), 10 mM MgCl 2 , 1 mM NAD + , 10 mM DTT, 60 mg/mL histone H1, 60 mg/mL histone type IIa, 50 mg/mL octameric oligonucleotide GGAATTCC, and 150 nM human PARP-1.…”