Characterizing the role of DDX41 in opposing transcription-associated genomic instability
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Abstract
Cycling of cells requires duplication of the genetic information which is encoded in the DNA. During the S-phase of the cell cycle, when DNA replication occurs, cells have to tolerate different types of replication stress. Lesions within the DNA template, DNA secondary structures, or the transcription machinery - through the formation of R-loops - can constitute obstacles for progressing replication forks. Conflicts between transcription-dependent R-loops, which are three-stranded structures consisting of an RNA-DNA hybrid and a displaced non-template DNA strand, and the replication machinery pose a threat to genomic stability. Moreover, R-loops regulate crucial cellular processes such as transcription initiation and termination, chromosome segregation, and DNA repair. Thus, R-loop levels need to be tightly regulated to maintain the regulatory functions as well as genome stability. Loss of R-loop homeostasis due to different cellular perturbations is associated with neurological disorders and cancer. The protein networks that regulate R-loops in human cells are poorly characterized. To this end, we developed RNA-DNA Proximity Proteomics (RDProx) to elucidate the regulatory R-loop proximal proteome in human cells.
Among different nuclear proteins associated with R-loops, we identified the tumor suppressor DEAD-box helicase DDX41. We show that DDX41 can bind and unwind RNA-DNA hybrids in vitro. Moreover, DDX41 associates with promoter regions of active genes to maintain R-loop homeostasis and to counteract the accumulation of DNA double-strand breaks in vivo. Loss of DDX41 results in R-loop-dependent DNA double-strand breaks, replication stress, and dependency on ATR signaling. R-loop accumulation is accompanied by changes in transcription dynamics and upregulation of inflammatory signature genes.
Germline and somatic mutations in DDX41 predispose patients to myelodysplastic syndromes and acute myeloid leukemia. Wild type DDX41 opposes DNA damage in CD34+ hematopoietic stem and progenitor cells, while expression of pathogenic variants leads to genomic instability. We propose that loss of promoter-proximal R-loop homeostasis, accumulation of DNA double-strand breaks, and inflammatory signaling contribute to the development of acute myeloid leukemia in individuals with pathogenic DDX41 variants.