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Interaction of Poly(ADP-ribose) and Specific Binding Proteins as a Function of Chain Length

Interaction of Poly(ADP-ribose) and Specific Binding Proteins as a Function of Chain Length

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FAHRER, Jörg, 2007. Interaction of Poly(ADP-ribose) and Specific Binding Proteins as a Function of Chain Length

@phdthesis{Fahrer2007Inter-6594, title={Interaction of Poly(ADP-ribose) and Specific Binding Proteins as a Function of Chain Length}, year={2007}, author={Fahrer, Jörg}, address={Konstanz}, school={Universität Konstanz} }

deposit-license 2007 Poly(ADP-ribosyl)ation is one of the very early cellular responses to genotoxic insults and is catalyzed by the family of poly(ADP-ribose) polymerases (PARPs). Using NAD+ as substrate, PARPs synthesize the biopolymer poly(ADP-ribose) (PAR), comprising up to 200 ADP-ribose units (Burkle 2005). This complex biopolymer interacts with a number of proteins involved in DNA damage checkpoint and repair via a specific consensus motif (Pleschke et al. 2000).<br />Objective of the thesis was to characterize the noncovalent interaction between sizefractionated PAR and specific binding partners such as p53 in terms of selectivity and affinity. PARP-1, the xeroderma pigmentosum-A [XPA] protein, p53 and WRN were overexpressed in Sf9 insect cells using the baculovirus system and purified to homogeneity. PAR was synthesized in vitro and efficiently biotin-labeled at the terminal ribose using the carbonylreactive compound biocytin hydrazide. Following anion exchange HPLC fractionation, the fractions collected were monitored for chain length on modified sequencing gels by silver staining and revealed isolated PAR chains ranging from 6 up to 70 ADP-ribose units. Interaction of separated PAR chains and recombinant proteins were studied by slot blot analysis and a novel electrophoretic mobility shift assay (EMSA). Moreover, real-time surface plasmon resonance (SPR) was used to assess binding kinetics and stoichiometry.<br />Slot blot experiments, which monitored the binding of PAR of a defined size class to immobilized proteins, clearly indicated a pivotal role for PAR chain length. EMSA studies were performed to monitor the binding affinities in solution. Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16mer) were also able to bind p53, yet forming only one defined complex. By contrast, XPA did not interact with short polymer, but did produce a single complex with long PAR chains (55mer). In the present work, the oncoprotein DEK was identified as a novel member of the PARbinding protein family. Like XPA, DEK underwent complex formation with long PAR chains, however with much higher affinity, but did not interact with short PAR. Finally, SPR analysis was carried out with immobilized PAR chains, which allowed establishing binding constants. In line with the EMSA experiments XPA did not bind to short PAR (14mer), but displayed a high affinity for long PAR chains (63mer), whereas p53 interacted strongly with both short and long PAR chains.<br />In summary, it was demonstrated that the affinity of the noncovalent PAR interaction with specific binding proteins (DEK, XPA, p53) can be very high (low nM range) and is dependent both on the PAR chain length and on the binding protein. These findings provide evidence for the existence of a cellular "PAR code", i.e. the ability of PAR to engage in different cellular signaling pathways as a function of PAR chain length. Interaction of Poly(ADP-ribose) and Specific Binding Proteins as a Function of Chain Length 2011-05-04T10:02:09Z Fahrer, Jörg Interaktion von Poly(ADP-Ribose) und spezifischen Bindeproteinen als Funktion der Kettenlänge Fahrer, Jörg eng application/pdf

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

Diss_Fahrer.pdf 173

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