Dual and opposing roles of the EXD2 exonuclease in the resolution of RNA-DNA hybrids.

Nucleases are specialized enzymes known for degrading nucleic acids in diverse cellular processes. Among them, EXD2 contributes to genome maintenance by digesting a broad range of nucleic acid substrates, including non-canonical RNA-DNA hybrids (RDHs). However, the molecular mechanism underlying the interplay of EXD2 with these hybrid structures has remained elusive. Here, using an optical tweezers-based single-molecule approach, we unveil that EXD2 cooperatively binds to and slowly digests mechanically tensioned RDHs. On the other hand, the cooperative binding of a high amount of EXD2 onto a relaxed RDH drives their co-condensation. Moreover, EXD2 is capable of recognizing damage sites along RDHs and inducing damaged RDH condensation even at low protein concentrations. Surprisingly, this co-condensation, in contrast, protects RDHs from timely degradation by other nucleases. Consistently, we unveil that overexpressed EXD2 colocalizes with mitochondrial RDHs and, rather than cleavage, prevents them from fast degradation. Therefore, EXD2 is a promiscuous moonlighting enzyme that can exert opposing activities toward RDHs. Our findings provide a mechanistic understanding of the functional roles of EXD2 in cells.