Stefán Sigurðsson, Karen Kristjánsdóttir, Birta Dröfn Jónsdóttir, Linda Viðarsdóttir and Þorkell Guðjónsson
Introduction: DNA double strand breaks (DSB) are the most cytotoxic DNA lesions the human genome endures and if left unrepaired, DSB can result in cell death. Incorrectly repaired DSB can result in the accumulation of mutation and chromosomal translocation an early step in the aetiology of carcinogenesis. ALKBH3 & FTO are dioxygenases involved in DNA/RNA alkylation repair and mRNA demethylation. We have shown that ALKBH3 and FTO play an important role in DNA-DSB repair by influencing RNF168, a key regulator of DNA-DSB repair. These enzymes impact RNF168 mRNA nuclear export by removing methylation marks from the RNF168-transcript.
Methods: We performed cell survival assays and assays to measure genetic instability in ALKBH3 and FTO knockout cell lines as well as siRNA treated cells. Mass spectrometry was also performed after immunoprecipitation using FLAG tagged versions of ALKHB3 and FTO.
Results: Cells lacking ALKBH3 or FTO show strong signs of RNF168 dysfunction, including genome instability and hypersensitivity to genotoxic agents. The mass spectrometry data revealed that ALKHB3 and FTO interact, supporting the functional interaction we have observed. In addition, novel binding partners of FTO and ALKHB3 were identified.
Conclusions: The increased drug sensitivity observed is likely to be explained by decreased recruitment of essential DNA-DSB repair proteins to the site of DNA damage which ultimately affects DNA repair. Due to both a lack of alkylation repair and defective DNA DSB repair, the deficiency of ALKBH3 and FTO may be used as potential markers for cancer treatment response or as potential treatment targets.
