MDS and AML associated DDX41 helicase resolves R-loops to maintain genome stability
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DNA damage and aberrant DNA damage response (DDR) are significant features of genomic instability that are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). DEAD-box helicase 41 (DDX41) is a member of helicase superfamily 2. Both germline and somatic mutations of DDX41 have been associated with MDS and AML, and missense mutant R525H is the most frequent mutation (67%) found in MDS and AML patients. However, the molecular pathogenesis of DDX41 mutations, such as R525H, remains unknown. Here, I found that DDX41 protein could efficiently unwind RNA:DNA hybrid in vitro. This prompted us to investigate the potential role of DDX41 in DDR concerning R-loops, one of the significant sources of genome instability. Examining DDX41 knockout (KO) cells (HeLa and HT1080) and DDX41 KO cells reconstituted with DDX41-R525H gene, I found that these cells had prolonged and increased DNA double-strand breaks (DSB) induced by ionizing radiation (IR) or bleomycin, a radiomimetic agent, compared to wildtype (WT) and DDX41 KO cells reconstituted with DDX41-WT gene, suggesting that DDX41 is crucial for DDR. Using the R-loop specific antibody S9.6, dot blot assays showed increased R-loops in DDX41-KO and R525H-expressing cells, deciphering that DDX41 functions as an R-loop resolvase. Furthermore, biochemical assays demonstrated R525H stimulates R-loops formation. Lastly, I found that DDX41 colocalized with the DSB marker γH2AX, R-loop marker S9.6, and transcription machinery marker RNA polymerase II, indicating that DDX41 interacts with proteins during transcription and DNA repair. In conclusion, our results imply that DDX41 is required for DDR as an R-loop resolvase upon DNA damage, and lack of or impaired DDX41 results in altered DDR response and pathogenic R-loop accumulation, which ultimately leads to genome instability and possibly pathogenesis of MDS and AML.
DegreeMaster of Science (M.Sc.)
SupervisorWu, Dr. Yuliang
CommitteeLeary, Dr. Scot; Lee, Dr. Jeremy; Zhou, Dr. Yan
Copyright DateDecember 2021
Helicase, DNA damage, R-loop