WRAP53β and scaRNA2 on their journey to repair the DNA

Abstract: WRAP53β is a multifunctional protein involved in several cellular processes like Cajal body formation, telomere maintenance and DNA repair. WRAP53β is responsible for targeting factors to specific cellular locations, and its very high affinity for the scaRNAs could implicate these molecules in similar biological functions. In Paper I we established a new function of WRAP53β protein in DNA damage response. We showed that WRAP53β localizes rapidly at sites of DNA double strand breaks (DSBs) in an ATM, ATR, H2AX and MDC1 dependent manner. WRAP53β acts as a scaffold for the interaction between the E3 ligase RNF8 and the upstream repair factor MDC1. Subsequently, RNF8 initiates an ubiquitin cascade that triggers the accumulation of downstream repair proteins like 53BP1, BRCA1, RAD51 at the break site. Loss of WRAP53β inhibits both repair pathways (homologous recombination and non-homologous end joining) and this leads to accumulation of spontaneous DNA damage and ultimately to cell death. In Paper II we introduced the proximity ligation assay (PLA), as a method for visualizing and analyzing factors that are recruited and form complexes at sites of DNA damage. By employing PLA we detected accumulation of WRAP53β in close proximity to γH2AX in an ATM, ATR dependent manner, a result we could also confirm by co-immunoprecipitation. With PLA we were able to observe the interaction of MDC1 and RNF8 that was abolished after depletion of WRAP53β or ATM inhibition. Finally, we monitored the phosphorylation of MDC1 that remained unaffected upon WRAP53β down-regulation. In Paper III we showed that WRAP53β protein is phosphorylated in serine64 (pWRAP53βs64) by ATM kinase in response to various types of DNA damage. Interestingly, pWRAP53βs64 is recruited to sites of DNA lesions and promotes its interaction with γH2AX. Furthermore, it stimulates the recruitment of the downstream factor 53BP1 and enhances repair with both HR and NHEJ pathways. In Paper IV we identified the C/D box scaRNA2 as an important player of the DNA damage response. ScaRNA2 is recruited directly to sites of DNA damage and promotes the HR repair pathway by facilitating the recruitment of the HR repair factors at the DSBs. ScaRNA2 seems to act in combination with the C/D box RNP complex, as members of this complex accumulate also at sites of DNA lesions. The catalytic domain of this complex belongs to the methyltransferase fibrillarin. Interestingly, depletion of fibrillarin impairs the recruitment of specific repair factors and reduces the efficiency of HR repair. To sum up, our data identify the Cajal body components WRAP53β and scaRNA2 as key regulators of the DNA repair process.