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