title: A quantitative assay reveals ligand specificity of the DNA scaffold repair protein XRCC1 and efficient disassembly of complexes of XRCC1 and the poly(ADP-ribose) polymerase 1 by poly(ADP-ribose) glycohydrolase.
abstract: The posttranslational modification of proteins with poly(ADP-ribose) (PAR) regulates protein-protein interactions in DNA repair, gene expression, chromatin structure, and cell fate determination. The PAR polymerase PARP1 binds to damaged chromatin and synthesizes PAR chains to signal DNA damage and recruit the DNA repair scaffold, XRCC1. Pharmacological blockade of PARP1 enzymatic activity impairs XRCC1-dependent repair of DNA damage and selectively kills cancer cells lacking other DNA repair functions. As such, PARP inhibitors are promising new therapies for repair-deficient tumors such as BRCA mutated breast cancers. Although the XRCC1-PARP1 complex is relevant to the proposed therapeutic mechanism of PARP inhibitors, the physical makeup and dynamics of this complex are not well characterized at the molecular level. Here we describe a fluorescence-based, real-time assay that quantitatively monitors interactions between PARylated PARP1 and XRCC1. Using this assay, we show that the PAR posttranslational modification by itself is a high affinity ligand for XRCC1, requiring a minimum chain length of 7 ADP-ribose units in the oligo(ADP-ribose) ligand for a stable interaction with XRCC1. This discrete binding interface enables the PAR glycohydrolase (PARG) to completely disassemble the PARP1-XRCC1 complex without assistance from a mono(ADP-ribose) glycohydrolase. Our quantitative, real-time assay of PAR-dependent protein-protein interactions and PAR turnover by PARG is an excellent tool for high-throughput screening to identify pharmacological modulators of PAR metabolism that may be useful therapeutic alternatives to PARP inhibitors.
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 inkwon@biochem.wustl.edu.
authors.forename: In-Kwon
authors.initials: IK
authors.lastname: Kim
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Roderick A
authors.initials: RA
authors.lastname: Stegeman
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Chris A
authors.initials: CA
authors.lastname: Brosey
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 tome@biochem.wustl.edu.
authors.forename: Tom
authors.initials: T
authors.lastname: Ellenberger
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006801:Humans; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D004268:DNA-Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D000246:Adenosine Diphosphate Ribose; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase
keywords: ADP-ribosylation; DNA Repair; Fluorescence Resonance Energy Transfer (FRET); High-throughput Screening (HTS); Hydrolase; Poly(ADP-ribose) Turnover; Poly(ADP-ribose)ylation; Posttranslational Modification (PTM); Time-resolved Fluorescence Resonance Energy Transfer