An in vivo Interaction Network of DNA-Repair Proteins: A Snapshot at Double Strand Break Repair in Deinococcus radiodurans.

Abstract

An extremophile Deinococcus radiodurans survives massive DNA damage by efficiently mending hundreds of double strand breaks through homology-dependent DNA repair pathways. Although DNA repair proteins that contribute to its impressive DNA repair capacity are fairly known, interactions among them or with proteins related to other relevant pathways remain unexplored. Here, we report in vivo cross-linking of the interactomes of key DNA repair proteins DdrA, DdrB, RecA, and Ssb (baits) in D. radiodurans cells recovering from gamma irradiation. The protein-protein interactions were systematically investigated through co-immunoprecipitation experiments coupled to mass spectrometry. From a total of 399 proteins co-eluted with the baits, we recovered interactions among diverse biological pathways such as DNA repair, transcription, translation, chromosome partitioning, cell division, antioxidation, protein folding/turnover, metabolism, cell wall architecture, membrane transporters, and uncharacterized proteins. Among these, about 80 proteins were relevant to the DNA damage resistance of the organism based on integration of data on inducible expression following DNA damage, radiation sensitive phenotype of deletion mutant, etc. Further, we cloned ORFs of 23 interactors in heterologous E. coli and expressed corresponding proteins with N-terminal His-tag, which were used for pull-down assays. A total of 95 interactions were assayed, in which we confirmed 25 previously unknown binary interactions between the proteins associated with radiation resistance, and 2 known interactions between DdrB and Ssb or DR_1245. Among these, five interactions were positive even under non-stress conditions. The confirmed interactions cover a wide range of biological processes such as DNA repair, negative regulation of cell division, chromosome partitioning, membrane anchorage, etc., and their functional relevance is discussed from the perspective of DNA repair. Overall, the study substantially advances our understanding on the cross-talk between different homology-dependent DNA repair pathways and other relevant biological processes that essentially contribute to the extraordinary DNA damage repair capability of D. radiodurans. The data sets generated and analyzed in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD021822.