DNA:RNA hybrids are more stable than double stranded DNA. In cells, considerable effort is undertaken during transcription to ensure that RNA does not anneal to its template, forming so-called “R-loops”. However, physiological or aberrant stalling of RNA polymerases can lead to formation of persistent R-loops that are a threat to genome stability. This is because the displaced ssDNA within an R-loop is prone to (1) cleavage by nucleases, creating DNA breaks, (2) recombination with distant DNA sequences, creating chromosome rearrangements, and (3) atypical modification by ssDNA viral defence proteins such as APOBEC enzymes. Enzymes that suppress R-loop formation would therefore be expected to maintain genome stability and suppress mutagenesis and cancer.
We have created a biochemical reconstitution system for in vitro investigation of R-loop processing. We show that a class of enzymes called DNA branchpoint translocases are highly efficient at displacement of RNA from such co-transcriptional R-loops. Branchpoint translocase enzymes are mutated in human disease and include FANCM (causative in the cancer predisposition syndrome Fanconi Anemia), SMARCAL1 (causative in Schimke immuno-osseous dysplasia) and ZRANB3 (strongly associated with type 2 diabetes). Surprisingly, other dsDNA helicases lack the ability to act on R-loops, suggesting that branchpoint translocation is a major mechanism of R-loop removal. Consistent with this biochemical activity, we show that FANCM localizes to regions of the genome that then accumulate DNA:RNA hybrids in FANCM knockout cells. FANCM, SMARCAL1 and ZRANB3 depletion causes a combined viability deficit due to elevated R-loop levels and increased cell cycle arrest. As predicted, genome instability is increased due to transcription-dependent DNA breakage, increased recombination and increased APOBEC modification of DNA. Our work reveals a mechanistic basis for R-loop displacement that is critical for genome stability.