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

Fahrer J, Kranaster R, Altmeyer M, Marx A, Bürkle A - Nucleic Acids Res. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Molecular Toxicology Group, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany.

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.

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Interaction of fractionated PAR and binding proteins in solution as assessed by EMSA. Briefly, biotinylated PAR of a defined size was incubated with binding proteins and subjected to native PAGE followed by semi-dry blotting. Bound and free ADP-ribose chains were detected using streptavidin–POD. (A) Binding of short PAR chains (16-mer) to XPA. (B) Binding of long PAR chains (55-mer) to XPA. (C) Quantitative evaluation of XPA gel shifts. Shift (%) was calculated as follows: signal intensity complexed PAR/(complexed + free PAR). Data are expressed as mean + SEM of triplicates from two independent experiments. (D) Binding of short PAR chains (16-mer) to p53. (E) Binding of long PAR chains (55-mer) to p53. (F) Evaluation of p53 gel shifts as described in (C).
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Figure 3: Interaction of fractionated PAR and binding proteins in solution as assessed by EMSA. Briefly, biotinylated PAR of a defined size was incubated with binding proteins and subjected to native PAGE followed by semi-dry blotting. Bound and free ADP-ribose chains were detected using streptavidin–POD. (A) Binding of short PAR chains (16-mer) to XPA. (B) Binding of long PAR chains (55-mer) to XPA. (C) Quantitative evaluation of XPA gel shifts. Shift (%) was calculated as follows: signal intensity complexed PAR/(complexed + free PAR). Data are expressed as mean + SEM of triplicates from two independent experiments. (D) Binding of short PAR chains (16-mer) to p53. (E) Binding of long PAR chains (55-mer) to p53. (F) Evaluation of p53 gel shifts as described in (C).

Mentions: To further analyze the non-covalent interaction of PAR and binding proteins as a function of chain length, a PAR EMSA was established. Increasing concentrations of recombinant XPA were incubated with fixed amounts of avidin affinity-purified short ADP-ribose chains (16-mer) and samples were subjected to native PAGE. Free as well as bound PAR polymer was detected via the terminal biotin-label using streptavidin–POD (Figure 3A). No specific interaction of XPA with short ADP-ribose chains was observed. At high XPA concentrations apparently some binding of PAR did occur, however without formation of a defined complex. Using long PAR chains (55-mer) XPA produced a complex in a concentration-dependent manner (Figure 3B). A concentration of 0.6 μM XPA was sufficient for complete binding of the available free polymer. Densitometric evaluation of the blots is depicted in Figure 3C, displaying the significant difference in PAR binding. In contrast, p53 promoted specific complex formation with short-PAR chains starting at 0.1 μM of p53 (Figure 3D). PAR was almost completely bound at 0.8 μM p53, as was detected by the formation of one discrete complex appearing at the top of the gel. The same set of experiments was repeated using long ADP-ribose molecules with an average chain length of 55 units (Figure 3E). Binding was detected already at or above 0.1 μM p53 and was nearly complete at 0.2 μM demonstrating the higher affinity of p53 for long chains. Strikingly, we observed that p53 was able to form three distinct specific complexes with long-PAR chains at higher concentrations. Blots were quantified and summarized clearly indicating the different affinities of p53 with regard to chain length (Figure 3F). Since all measurements were made in equilibrium, the EMSA results allowed the calculation of KD values representing the affinities of the respective proteins to the isolated PAR chains. The KD values we determined were all in the nanomolar range, demonstrating the high affinity of this non-covalent interaction (Table 1; Supplementary Table S1). In addition, there exists a binding specificity with regard to chain length as XPA was not able to form a specific complex with short ADP-ribose molecules in solution.Figure 3.


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

Fahrer J, Kranaster R, Altmeyer M, Marx A, Bürkle A - Nucleic Acids Res. (2007)

Interaction of fractionated PAR and binding proteins in solution as assessed by EMSA. Briefly, biotinylated PAR of a defined size was incubated with binding proteins and subjected to native PAGE followed by semi-dry blotting. Bound and free ADP-ribose chains were detected using streptavidin–POD. (A) Binding of short PAR chains (16-mer) to XPA. (B) Binding of long PAR chains (55-mer) to XPA. (C) Quantitative evaluation of XPA gel shifts. Shift (%) was calculated as follows: signal intensity complexed PAR/(complexed + free PAR). Data are expressed as mean + SEM of triplicates from two independent experiments. (D) Binding of short PAR chains (16-mer) to p53. (E) Binding of long PAR chains (55-mer) to p53. (F) Evaluation of p53 gel shifts as described in (C).
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Figure 3: Interaction of fractionated PAR and binding proteins in solution as assessed by EMSA. Briefly, biotinylated PAR of a defined size was incubated with binding proteins and subjected to native PAGE followed by semi-dry blotting. Bound and free ADP-ribose chains were detected using streptavidin–POD. (A) Binding of short PAR chains (16-mer) to XPA. (B) Binding of long PAR chains (55-mer) to XPA. (C) Quantitative evaluation of XPA gel shifts. Shift (%) was calculated as follows: signal intensity complexed PAR/(complexed + free PAR). Data are expressed as mean + SEM of triplicates from two independent experiments. (D) Binding of short PAR chains (16-mer) to p53. (E) Binding of long PAR chains (55-mer) to p53. (F) Evaluation of p53 gel shifts as described in (C).
Mentions: To further analyze the non-covalent interaction of PAR and binding proteins as a function of chain length, a PAR EMSA was established. Increasing concentrations of recombinant XPA were incubated with fixed amounts of avidin affinity-purified short ADP-ribose chains (16-mer) and samples were subjected to native PAGE. Free as well as bound PAR polymer was detected via the terminal biotin-label using streptavidin–POD (Figure 3A). No specific interaction of XPA with short ADP-ribose chains was observed. At high XPA concentrations apparently some binding of PAR did occur, however without formation of a defined complex. Using long PAR chains (55-mer) XPA produced a complex in a concentration-dependent manner (Figure 3B). A concentration of 0.6 μM XPA was sufficient for complete binding of the available free polymer. Densitometric evaluation of the blots is depicted in Figure 3C, displaying the significant difference in PAR binding. In contrast, p53 promoted specific complex formation with short-PAR chains starting at 0.1 μM of p53 (Figure 3D). PAR was almost completely bound at 0.8 μM p53, as was detected by the formation of one discrete complex appearing at the top of the gel. The same set of experiments was repeated using long ADP-ribose molecules with an average chain length of 55 units (Figure 3E). Binding was detected already at or above 0.1 μM p53 and was nearly complete at 0.2 μM demonstrating the higher affinity of p53 for long chains. Strikingly, we observed that p53 was able to form three distinct specific complexes with long-PAR chains at higher concentrations. Blots were quantified and summarized clearly indicating the different affinities of p53 with regard to chain length (Figure 3F). Since all measurements were made in equilibrium, the EMSA results allowed the calculation of KD values representing the affinities of the respective proteins to the isolated PAR chains. The KD values we determined were all in the nanomolar range, demonstrating the high affinity of this non-covalent interaction (Table 1; Supplementary Table S1). In addition, there exists a binding specificity with regard to chain length as XPA was not able to form a specific complex with short ADP-ribose molecules in solution.Figure 3.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Molecular Toxicology Group, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany.

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.

Show MeSH
Related in: MedlinePlus