Functional characterization of the RNF169-DYRK1A complex in DNA double-strand break signaling and repair

Ubiquitylation at DNA double-strand breaks (DSBs) plays a critical role in the orchestration of DNA damage signaling and repair. However, it remains obscure how ubiquitylation reactions and the resulted products are restrained at DSBs. With a bioinformatics approach, the RING finger protein RNF169 was identified as a protein that bears a similar domain organization to RNF168, which is a pivotal E3 ligase that promotes the ubiquitin-mediated DSB response. Upon challenge by ionizing radiation (IR)-induced genotoxic stress, RNF169 migrated to DSBs and docked at RNF168-promoted ubiquitin conjugates. RNF169 competed for ubiquitin conjugates at DSBs; displaced DNA damage signaling and repair proteins, including RNF168 from DNA lesions; and relieved cell cycle arrest at the G2/M checkpoint, indicative of negative roles in the ubiquitin-mediated DSB signaling cascade. However, the cellular role of RNF169, and how RNF169 is regulated remain elusive. To better appreciate how RNF169 restrains DSB ubiquitylation and signaling events, RNF169 protein complexes were purified, and dual specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was identified as an RNF169-interacting partner. DYRK1A accumulated at DSBs through its interaction with RNF169 and was crucial for cell resistance to IR-induced genotoxic stress, suggesting a role of DYRK1A in the DNA damage response. In agreement with the negative role of its interacting partner RNF169, ectopically expressed DYRK1A limited the assembly of DNA damage response proteins at neighboring DSBs via its kinase activity. Conversely, ablation of DYRK1A markedly elevated spontaneous p53 binding protein 1 (53BP1) foci, suggesting that DYRK1A might regulate chromatin accessibility. Indeed, depletion of DYRK1A not only significantly promoted DSB repair but also sensitized chromatin to micrococcal nuclease (MNase) digestion. Furthermore, my preliminary data also showed that DYRK1A readily phosphorylated histone H3 at threonine 45, a mark that alters nucleosome stability and conformation. In conclusion, we propose that DYRK1A remodels chromatin at DSB domains, and that the RNF169-DYRK1A complex employs two different mechanisms: competitive inhibition and reduced chromatin accessibility, to limit DNA damage signal from over-spreading on DSB chromatin regions.published_or_final_versionBiomedical SciencesDoctoralDoctor of Philosoph