Sensoy, O.; Carvajal-Garcia, J.; Heyza, J.; Wiggins, P.; Merrikh, H.

RNA:DNA hybrids form across all genomes. In vitro studies showed that these structures can be used to restart stalled replication forks, especially upon replication-transcription conflicts, however, this has not been tested in vivo. Here, we identify a mechanism that prevents replication restart from hybrids within cells. We identified the tRNA ligase, AsnRS, as a regulator of replication restart from hybrids at conflict regions in Bacillus subtilis. We determined that the DNA translocase Mfd and DNA polymerase PolA play key roles in this pathway. We unraveled a mechanism whereby Mfd removes RNA Polymerases, exposing 3' ends of the RNAs within hybrids. These 3' ends are then capped by AsnRS, preventing restart by PolA. Remarkably, the mammalian homologs of AsnRS, NARS1 and NARS2, fully complement the AsnRS phenotypes in bacteria. We propose that this is a universally conserved mechanism that prevents untimely replication initiation outside of origins from bacteria to humans.