title: The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1.
abstract: We recently demonstrated that WRAP53β acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53β accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immunoprecipitation (IP) binds to γH2AX, in a manner dependent on the ATM and ATR kinases. Moreover, formation of complexes between MDC1 and both its partners RNF8 and phosphorylated ATM was visualized. The interaction of MDC1 with RNF8, but not with ATM requires WRAP53β, suggesting that WRAP53β facilitates the former interaction without altering phosphorylation of MDC1 by ATM. Furthermore, our findings highlight PLA as a more sensitive method for the analysis of recruitment of repair factors and complex formation at DNA breaks that are difficult to detect using conventional immunofluorescence.
authors.affiliation: a Department of Oncology-Pathology ; Cancer Centrum Karolinska (CCK); Karolinska Institutet ; Stockholm , Sweden.
authors.forename: Hanif
authors.initials: H
authors.lastname: Rassoolzadeh
authors.affiliation: a Department of Oncology-Pathology ; Cancer Centrum Karolinska (CCK); Karolinska Institutet ; Stockholm , Sweden.
authors.forename: Christos
authors.initials: C
authors.lastname: Coucoravas
authors.affiliation: a Department of Oncology-Pathology ; Cancer Centrum Karolinska (CCK); Karolinska Institutet ; Stockholm , Sweden.
authors.forename: Marianne
authors.initials: M
authors.lastname: Farnebo
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011485:Protein Binding; D019098:Telomerase / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C413163:MDC1 protein, human; D018832:Molecular Chaperones; D009687:Nuclear Proteins; C486521:RNF8 protein, human; D015534:Trans-Activators; D044767:Ubiquitin-Protein Ligases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D019098:Telomerase; C535133:WRAP53 protein, human
keywords: ATM; DNA damage response; Double-strand breaks; MDC1; PLA; RNF8; TCAB1; WDR79; WRAP53; γH2AX