Abstract B10: The role of Down syndrome's DYRK1A kinase in repair of the DNA double strand breaks

Abstract The function of DYRK1A protein kinase is regulated by its gene dosage whereby both gains and losses of one copy of DYRK1A gene on chromosome 21 result in developmental abnormalities. In order to better understand the function and regulation of DYRK1A, we applied a highly sensitive MudPIT proteomic approach to identify DYRK1A-interacting proteins in human cells. Four biological replicate MudPIT experiments were performed and the proteins reproducibly detected in the DYRK1A immunoprecipitates but not in the controls, were identified. Six proteins detected in all four biological replicate experiments were also most highly enriched in the DYRK1A immunoprecipitates, suggesting that these proteins form stable and abundant complexes with DYRK1A. One of these proteins, RNF169, has been recently characterized as a component of ubiquitin-mediated cascade involved in the repair of DNA double-strand breaks (DSBs). DSBs are deleterious DNA lesions that are repaired by hierarchical and orchestrated recruitment of multiple different proteins to a modified chromatin in the vicinity of the DNA damage sites. Presence of specific chromatin marks including ubiquitination regulates the choice between two major DSB repair pathways: homologous recombination repair (HRR) and non-homologous end joining (NHEJ), mediated by recruitment of chromatin-binding DNA damage response proteins including 53BP1 and RNF169. Binding of 53BP1 could prevent the resection of the DNA strands near the damage site necessary for the HRR while RNF169 is thought to limit the 53BP1 accumulation and therefore, to promote the HRR. To determine whether DYRK1A plays a role in these processes, we knocked out its expression in human and mouse cell lines using CRISPR-Cas9 approach. We found that initial accumulation of 53BP1 at the gamma-irradiation induced foci (IRIFs) was similar in the DYRK1A-null and in the control cells. However, both the number of the 53BP1 IRIFs and their persistence over time were significantly reduced in the cell lines that lacked DYRK1A. This effect was dependent on the presence of RNF169, suggesting that DYRK1A regulates the ability of RNF169 to limit 53BP1's accumulation at the DSBs. Next, we sought to determine the mechanism of this regulation and found that RNF169 is phosphorylated by DYRK1A at two sites located in a highly conserved domain with no known function. Interestingly, the phospho mimetic mutant of RNF169 displayed a decreased ability to inhibit 53BP1 IRIF formation when compared to the wild type or the non-phosphorylatable RNF169 alleles. We also determined the effect of DYRK1A overexpression on the recruitment of RNF169 and 53BP1 to the sites of IR-induced DNA damage. Surprisingly, accumulation of both RNF169 and 53BP1 in the IRIFs was diminished upon overexpression of active, but not the kinase-inactive DYRK1A. Domain-mapping of DYRK1A-RNF169 binding demonstrated that the ability of DYRK1A to abolish the RNF169 IRIF formation is independent of their interaction, suggesting that overexpression of functional DYRK1A could affect other factors in the cell that play a role in the DNA damage response. Since loss of DYRK1A could be relevant to cancer due to its widespread gene copy number losses, we determined the effect of DYRK1A loss on the ability of the cells to repair their DNA. Using the DR-GFP reporter of HRR and the neutral comet assays, we found that CRISPR-Cas9-mediated depletion of DYRK1A results in an increased efficiency of the DNA DSB repair. Our findings implicate DYRK1A in the critical processes of DNA damage response and characterize a novel function of this important protein kinase. Citation Format: Vijay R. Menon, Varsha Ananthapadmanabhan, Larisa Litovchick. The role of Down syndrome's DYRK1A kinase in repair of the DNA double strand breaks [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B10.

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