A New Proteomics Method Links Excess R-loops to DNA Double-Strand Breaks and Genomic Instability

Researchers at the Institute of Molecular Biology (IMB) in Mainz, Germany, have developed a new method to identify proteins that bind to RNA-DNA structures called R-loops. Using this method, they show that a protein called DDX41 is responsible for preventing R-loops from building up in cells and that this is an important mechanism for preventing genomic instability and cancer. The results were published in the journal Nature Communications.

Our DNA contains all the information necessary for cells to function. It normally exists as a double-stranded helix. So-called R-loops are structures formed when an RNA strand binds to one strand of the DNA, causing the other DNA strand to form a single-stranded 'loop.' These R-loops are important for regulating gene transcription but unfortunately also increase the risk of DNA damage, which can, in turn, result in genomic instability and lead to cancer.

To find out how cells balance R-loop formation to permit transcription while also minimizing the risk of DNA damage, a team of researchers at IMB comprising the Beli, Roukos, Luke, and Barau labs developed a new method called RNA-DNA proximity (RDPRox) proteomics to screen for proteins that associate with R-loops. This allowed them to identify proteins responsible for regulating R-loop levels in human cells.

The researchers identified a protein called DDX41, frequently mutated in patients with myelodysplastic syndrome and acute myeloid leukemia. When the researchers experimentally reduced DDX41 protein levels, they found a dramatic increase in R-loops and double-strand breaks (DSBs) in the cells' DNA, suggesting that DDX41 is responsible for keeping R-loop levels down. Further analysis showed that this increase in R-loops primarily occurred at the promoters of highly expressed genes and that many promoters with increased R-loops also showed an increase in DSBs.

The researchers speculate that the accumulated R-loops in DDX41 mutant cells may interfere with DNA replication or transcription, increasing the chances of DSBs and leading to cancer. Prof. Petra Beli, the lead scientist in the project, says, "By identifying proteins that are proximal to R-loops in human cells, we showed for the first time that sites of R-loop accumulation coincide with increased DNA fragility." These findings may also open new therapeutic strategies to treat cancer by inhibiting DNA repair proteins.

Further details

Further information can be found at https://www.nature.com/articles/s41467-021-27530-y.

Petra Beli is an Adjunct Director at IMB and a Professor of Biology at Johannes Gutenberg University Mainz. Further information about research in the Beli lab can be found at www.imb.de/beli.

Joan Barau is a Group Leader at IMB. Further information about research in the Barau lab can be found at www.imb.de/barau.

Brian Luke is an Adjunct Director at IMB and a Professor of Biology at Johannes Gutenberg University Mainz. Further information about research in the Luke lab can be found at www.imb.de/luke.

Vassilis Roukos is a Group Leader at IMB. Further information about research in the Roukos lab can be found at www.imb.de/roukos.