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MDS/AML-associated DDX41 helicase facilitates homologous recombination repair by potentially resolving R-loops
Aanchal Aggarwal1, Shizhuo Yang1, Lacey Winstone1
1Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada.
Abstract:
DDX41 mutations are associated with myeloid neoplasms, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), and missense mutant R525H is seen in 67% of patients; however, its molecular pathogenesis is unknown. Using DDX41 knockout (KO) cells, we found that these cells were sensitive to bleomycin, camptothecin, and UV. DDX41 deficiency led to increased genomic instability, indicated by elevated DNA double-strand breaks (DSBs) and comet tails. We found that R-loop formation increased in DDX41-KO cells. DDX41 wild-type (WT) protein resolved DNA:RNA hybrid of R-loops in vitro, but the mutant R525H failed. DDX41-R525H expressing cells were sensitive to DNA damage agents and had significantly more R-loops than DDX41-WT expressing cells. Interestingly, DDX41 colocalized with DSB marker γH2AX and R-loop marker S9.6, and knockdown of DDX41 in the U2OS GFP reporter cells resulted in reduced homologous recombination (HR) repair. Moreover, increased and prolonged RPA and reduced RAD51 foci were found in DDX41 KO and DDX41-R525H expressing cells, indicating a defect in the transition from end resection to RAD51 filament assembly. Overall, our results suggest that DDX41 utilizes its unwinding activity to resolve R-loops, which may play a key role in HR-based repair, and dysregulation of this pathway may lead to MDS/AML.
Insights
DDX41 mutations linked to MDS/AML impair DNA repair by failing to resolve R-loops. This genomic instability suggests DDX41
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- DDX41 mutations are linked to myeloid neoplasms like myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).
- The specific R525H missense mutant is prevalent in 67% of affected patients, but its pathogenic mechanism remains unclear.
- Understanding DDX41's role is crucial for elucidating the molecular basis of these hematological malignancies.
Purpose of the Study:
- To investigate the molecular pathogenesis of DDX41 mutations in myeloid neoplasms.
- To determine the function of DDX41 in maintaining genomic stability and DNA repair.
- To explore the role of the R525H mutant in cellular response to DNA damage.
Main Methods:
- Utilized DDX41 knockout (KO) cell lines to assess sensitivity to DNA damaging agents (bleomycin, camptothecin, UV).
- Quantified DNA double-strand breaks (DSBs), R-loop formation, and DNA repair pathway markers (γH2AX, RPA, RAD51, S9.6).
- Performed in vitro assays with wild-type (WT) and mutant R525H DDX41 proteins to assess DNA:RNA hybrid resolution.
Main Results:
- DDX41-KO cells exhibited increased sensitivity to DNA damage, elevated DSBs, and heightened R-loop accumulation.
- Wild-type DDX41 resolved DNA:RNA hybrids in vitro, whereas the R525H mutant failed to do so.
- DDX41 deficiency and R525H expression impaired homologous recombination (HR) repair, indicated by altered RPA and RAD51 foci.
Conclusions:
- DDX41 plays a critical role in resolving R-loops, thereby maintaining genomic stability and facilitating HR-based DNA repair.
- The R525H mutation disrupts DDX41's unwinding activity, leading to R-loop accumulation and defective DNA repair.
- Dysregulation of the DDX41-mediated R-loop resolution pathway contributes to the pathogenesis of MDS and AML.
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