Characterization of a pathway of genomic instability induced by R-loops and its regulation by topoisomerases in E. coli

Author summaryDNA topoisomerases act during replication, transcription, and recombination to solve topological problems inherent to the double-helical structure of DNA. Topos of the type 1A family are the only ones that are ubiquitous. The prototype enzymes of the two main type IA subfamilies are topo I and topo III from Escherichia coli. The recent finding that E. coli topo I and III can suppress R-loop formation suggested that R-loops have been a problem early in the evolution of life. However, how toxic R-loops are generated and how they exert their detrimental effect on genome stability, especially in the absence of type IA topos, is still largely unknown. Here, we have uncovered a pathway leading to genome instability in the absence of type IA topos: unregulated replication from R-loops involving RNAP backtracking leads to DNA amplification in the chromosome terminus region due to replication forks blocked at termination barriers. Furthermore, our data suggest that type IA topos play major roles at the termination step of replication when convergent replication forks merge and R-loops exert their detrimental effects mostly via topological problems. Overall, our data shed new light on how R-loops can lead to genomic instability and how topos can deal with this problem. The prototype enzymes of the ubiquitous type IA topoisomerases (topos) family are Escherichia coli topo I (topA) and topo III (topB). Topo I shows preference for relaxation of negative supercoiling and topo III for decatenation. However, as they could act as backups for each other or even share functions, strains lacking both enzymes must be used to reveal the roles of type IA enzymes in genome maintenance. Recently, marker frequency analysis (MFA) of genomic DNA from topA topB null mutants revealed a major RNase HI-sensitive DNA peak bordered by Ter/Tus barriers, sites of replication fork fusion and termination in the chromosome terminus region (Ter). Here, flow cytometry for R-loop-dependent replication (RLDR), MFA, R-loop detection with S9.6 antibodies, and microscopy were used to further characterize the mechanism and consequences of over-replication in Ter. It is shown that the Ter peak is not due to the presence of a strong origin for RLDR in Ter region; instead RLDR, which is partly inhibited by the backtracking-resistant rpoB*35 mutation, appears to contribute indirectly to Ter over-replication. The data suggest that RLDR from multiple sites on the chromosome increases the number of replication forks trapped at Ter/Tus barriers which leads to RecA-dependent DNA amplification in Ter and to a chromosome segregation defect. Overproducing topo IV, the main cellular decatenase, does not inhibit RLDR or Ter over-replication but corrects the chromosome segregation defect. Furthermore, our data suggest that the inhibition of RLDR by topo I does not require its C-terminal-mediated interaction with RNA polymerase. Overall, our data reveal a pathway of genomic instability triggered by R-loops and its regulation by various topos activities at different steps.