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 RNA, DNA, and RNA–DNA hybrids (RDHs). However, the molecular mechanism underlying EXD2’s interplay with non-canonical hybrid structures has remained elusive. Here, we find that elevated EXD2 colocalizes mitochondria RNA–DNA hybrids, and regulates them in a dual, dose-dependent switching manner. 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. Therefore, EXD2 is a promiscuous moonlighting enzyme that can exert opposing activities toward RDHs. Our findings provide a mechanistic understanding of EXD2’s diverse functional roles in cells. This dataset contains all raw experimental data presented in the main text of the manuscript. The files are organized into separate folders corresponding to main figures and supplementary figures.