Unwrapping the role of WRAP53β in DNA damage response

Sammanfattning: WRAP53β is a WD40 domain protein with multifaceted capabilities in several biological processes in different cellular compartments. This includes transportation of the Survival of motor neuron (SMN) complex, small Cajal body-specific (sca) RNAs and the telomerase enzyme to Cajal bodies, as well as targeting of the E3 ligase RNF8 to DNA double-strand breaks (DSBs). In Paper I, we established WRAP53β as a novel player in the DNA damage response. WRAP53β is rapidly recruited to DNA lesions in an ATM, ATR, H2AX and MDC1 dependent manner, and accumulates at the actual breaks and surrounding regions. WRAP53β interacts with the E3 ubiquitin ligase RNF8 and facilitates binding to MDC1 by simultaneously binding RNF8 and MDC1 via its WD40 domain. This is important for the downstream recruitment of the repair factors 53BP1, BRCA1 and RAD51. Knockdown of WRAP53β impairs DSB repair by both non-homologous end joining (NHEJ) and homologous recombination (HR), causing accumulation of DNA lesions and delayed recovery from G2/M cell cycle arrest. In Paper II, we introduced a sensitive method for analyzing the recruitment of repair factors and formation of protein-protein complexes at DNA lesions. By applying a fluorescence visualization technique called the proximity ligation assay (PLA), we observed accumulation of WRAP53β in close proximity to γH2AX in an ATM and ATR dependent manner, which we also confirmed by co-immunoprecipitation. Moreover, we visualized the interaction between MDC1 and RNF8 via PLA and confirmed that these interactions are dependent on WRAP53β and ATM. Our results also demonstrated that WRAP53β does not interfere with MDC1 phosphorylation by ATM but instead contributes directly to the MDC1 and RNF8 interaction. In Paper III, we studied the role of overexpressed WRAP53β in DSB repair. We have demonstrated that overexpression of WRAP53β disrupts Cajal body formation, and at the same time, enhances the efficiency of DSB repair by HR and NHEJ. Accordingly, γH2AX foci were cleared faster in irradiated cells overexpressing WRAP53β. We also observed increased ubiquitylation of histone H2AX and that this response was attenuated when the responsible enzyme RNF8 was knocked down. WRAP53β overexpressing cancer cells were also resistant to irradiation and several damage-inducing agents. Altogether, this indicates that overexpressed WRAP53β enhances RNF8-mediated ubiquitylation at DNA lesions and thereby increases the repair efficiency and improves cancer cell survival. In summary, we have identified WRAP53β as an essential player in the DNA damage response (DDR). We could verify its function at damage sites by using the PLA technique and furthermore showed overexpression of this protein enhances DNA repair efficiency.