R-loops are stable structures that form when RNA becomes trapped within duplex DNA. R-loops are important in regulation of cellular processes such as gene expression, telomere maintenance, immunoglobulin (Ig) class switching, and DNA repair. More recently, it has been shown that changes in R-loop regulation induce DNA damage and genome instability.
In particular, down-regulation of certain genes of the Fanconi Anaemia (FA) DNA repair pathway, results in an accumulation of R-loops. This suggests the FA pathway plays a role in R-loop metabolism and regulation [1, 2]. In particular, we have shown that the FA-associated gene FANCM can displace trapped RNA from an R-loop to maintain genome stability.
To further elucidate the mechanism by which the FANCM and the FA pathway regulates R-loop processing we have created a biochemical system for in vitro analysis of R-loops. We show that FANCM processively unwinds both short and long R-loops, irrespective of sequence, topology or coating by replication protein A. R-loops can also be unwound in the same assay by the yeast and bacterial orthologs of FANCM, Mph1 and RecG, indicating an evolutionary conserved function.
Consistent with this biochemical activity of FANCM, we show that FANCM deficient cells are sensitive to drugs that stabilize R-loop formation. Our work reveals a mechanistic basis for R-loop metabolism that is critical for genome stability.