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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 immobilized proteins. (A) Recombinant purified p53 was vacuum-aspirated onto a nitrocellulose membrane using a slot-blot manifold (15 pmol/slot). The membrane was cut into slices and incubated with PAR fractions comprising distinct polymer size classes. After several washing steps with high stringency to disrupt unspecific protein–polymer interactions, bound PAR was detected by monoclonal antibody 10H followed by incubation with goat α-mouse HRP and peroxidase reaction. A representative slot-blot with triplicate determinations is shown. (B) Comparison of PAR binding with regard to chain length between XPA and p53. Signal intensity is indicated in arbitrary units. The bars represent mean + SEM of triplicates. Note the superior binding capacity of XPA for 20–49-mers, compared to p53.
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Figure 2: Interaction of fractionated PAR and immobilized proteins. (A) Recombinant purified p53 was vacuum-aspirated onto a nitrocellulose membrane using a slot-blot manifold (15 pmol/slot). The membrane was cut into slices and incubated with PAR fractions comprising distinct polymer size classes. After several washing steps with high stringency to disrupt unspecific protein–polymer interactions, bound PAR was detected by monoclonal antibody 10H followed by incubation with goat α-mouse HRP and peroxidase reaction. A representative slot-blot with triplicate determinations is shown. (B) Comparison of PAR binding with regard to chain length between XPA and p53. Signal intensity is indicated in arbitrary units. The bars represent mean + SEM of triplicates. Note the superior binding capacity of XPA for 20–49-mers, compared to p53.

Mentions: To assess the non-covalent binding of fractionated PAR chains to proteins, a classical approach was chosen. Equal amounts of each purified protein were immobilized on a nitrocellulose membrane and bound ADP-ribose chains were detected using the monoclonal PAR antibody 10H. The human tumor suppressor protein p53 was previously shown to interact in a non-covalent fashion with PAR and harbors three potential PAR-binding sites (20). Since the binding of PAR to p53 influences its DNA-binding activity we asked whether there was specificity for a certain polymer size class. Surprisingly, ADP-ribose chains ranging from 5 up to 39 units bound rather poorly to p53 (Figure 2A) whereas longer PAR chains displayed a higher affinity. Furthermore, the binding of separated PAR chains to immobilized human XPA was monitored. Short chains of up to 20 ADP-ribose moieties displayed weak affinity for XPA similar to the experiments performed with p53 (Figure 2B). Interestingly, PAR chains of 20–40 units showed higher affinity to XPA, compared to p53. PAR with more than 40 ADP-ribose units bound very tightly to XPA, comparable to p53.Figure 2.


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 immobilized proteins. (A) Recombinant purified p53 was vacuum-aspirated onto a nitrocellulose membrane using a slot-blot manifold (15 pmol/slot). The membrane was cut into slices and incubated with PAR fractions comprising distinct polymer size classes. After several washing steps with high stringency to disrupt unspecific protein–polymer interactions, bound PAR was detected by monoclonal antibody 10H followed by incubation with goat α-mouse HRP and peroxidase reaction. A representative slot-blot with triplicate determinations is shown. (B) Comparison of PAR binding with regard to chain length between XPA and p53. Signal intensity is indicated in arbitrary units. The bars represent mean + SEM of triplicates. Note the superior binding capacity of XPA for 20–49-mers, compared to p53.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2175335&req=5

Figure 2: Interaction of fractionated PAR and immobilized proteins. (A) Recombinant purified p53 was vacuum-aspirated onto a nitrocellulose membrane using a slot-blot manifold (15 pmol/slot). The membrane was cut into slices and incubated with PAR fractions comprising distinct polymer size classes. After several washing steps with high stringency to disrupt unspecific protein–polymer interactions, bound PAR was detected by monoclonal antibody 10H followed by incubation with goat α-mouse HRP and peroxidase reaction. A representative slot-blot with triplicate determinations is shown. (B) Comparison of PAR binding with regard to chain length between XPA and p53. Signal intensity is indicated in arbitrary units. The bars represent mean + SEM of triplicates. Note the superior binding capacity of XPA for 20–49-mers, compared to p53.
Mentions: To assess the non-covalent binding of fractionated PAR chains to proteins, a classical approach was chosen. Equal amounts of each purified protein were immobilized on a nitrocellulose membrane and bound ADP-ribose chains were detected using the monoclonal PAR antibody 10H. The human tumor suppressor protein p53 was previously shown to interact in a non-covalent fashion with PAR and harbors three potential PAR-binding sites (20). Since the binding of PAR to p53 influences its DNA-binding activity we asked whether there was specificity for a certain polymer size class. Surprisingly, ADP-ribose chains ranging from 5 up to 39 units bound rather poorly to p53 (Figure 2A) whereas longer PAR chains displayed a higher affinity. Furthermore, the binding of separated PAR chains to immobilized human XPA was monitored. Short chains of up to 20 ADP-ribose moieties displayed weak affinity for XPA similar to the experiments performed with p53 (Figure 2B). Interestingly, PAR chains of 20–40 units showed higher affinity to XPA, compared to p53. PAR with more than 40 ADP-ribose units bound very tightly to XPA, comparable to p53.Figure 2.

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