Efficient processing of widespread R-loops through an endo- and exo-cleavage coupling mode prevents genome instability

Excessive accumulation of R-loops is thought to be one of the major causes of genome instability. Endonuclease RNaseH1 acts as a classic, common R-loop resolvase to prevent R-loop accumulation genome-widely, whether there is any exonuclease complementing and teaming up with RNaseH1 to more efficiently resolve genome-wide R-loops is worth investigation. Here we identify an exonuclease REXO4, which collaborates with RNaseH1 endonuclease to more efficiently degrade R-loops in an “endo- and exo-cleavage coupling” manner. Specifically, among many members of DEDDh exonuclease family, REXO4 has the most significant effect on R-loop suppression. REXO4 directly degrades RNA strand in R-loop from the end or internal nick through its 3’-5’ exonuclease activity, and also stimulates RNaseH1 endonuclease activity. The genome-wide R-loop regions regulated by REXO4 highly overlap with those regulated by RNaseH1, and REXO4 overexpression counteracts genome-wide R-loop accumulation caused by RNaseH1 deficiency. Furthermore, REXO4-deficient tumors display elevated R-loop mutation burden, and tumor patient-derived mutations in REXO4 enzymatic region all impair R-loop cleavage activity. Interfering with REXO4 increases the sensitivity of tumor cells to alkylating and G4 stabilizing drugs. Therefore, our study proposes a model for highly efficient processing of genome-wide R-loops by endo- and exo-cleavage, which provides additional insights into understanding the link between R-loop associated genome instability and tumors.