Roles of Human DDX47 Helicase in R-Loop Resolution




Amdeen, Shahad Adnan

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R-loops are physiological structures that serve important roles in gene regulation, DNA repair, and other cellular processes. R-loops can also be problematic to the cells and cause replication stress as well as DNA strand breaks which lead to genomic instability. Therefore, cells have developed methods for preventing the formation of R-loops, or resolving the pathological R-loops, but the detailed mechanism of R-loop resolution by different repair factors remains unclear. One of these methods to prevent R-loop accumulation is by DNA/RNA helicases which can unwind DNA-RNA hybrids or R-loops. Dysfunction of such DNA/RNA helicases usually causes R-loop accumulation, leading to replication stress, DNA single- or double-strand breaks, genome instability and cancer susceptibility. Excessive R-loops are a characteristic of several neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Fragile X syndrome and Friedreich’s ataxia. Our preliminary data showed that depletion of DDX47, a human RNA helicase functioning in nuclear and mitochondrial splicing, could cause accumulation of R-loops in human HeLa cells indicating that DDX47 was involved in R-loop resolution in cells. In this project, we purified wild type human DDX47 as well as its helicase impaired mutants K74A and E175A. Using these purified proteins, we were able to demonstrate that only wild type DDX47, but not its helicase impaired mutants, could unwind dsDNA, DNA-RNA hybrids as well as RNA-RNA duplexes in vitro. We also found that DDX47 was able to dissociate a modeled R-loop structure in vitro, which was also dependent on its helicase activity. Finally, purified DDX47 was found to bind to R-loop structures via EMSA assays. Our results helped us to understand the function of DDX47 in R-loop resolution, which may lead to possible therapeutic remedies for human cancer and neurodegenerative disorders caused by pathological R-loop accumulation.



r-loops, DDX47, electrophoretic mobility shift assay, EMSA, DNA


Amdeen, S. A. (2023). Roles of human DDX47 helicase in R-loop resolution (Unpublished thesis). Texas State University, San Marcos, Texas.


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