Synthetic lethal mutants in Escherichia coli define pathways necessary for survival with RNase H deficiency

Ribonucleotides frequently contaminate DNA and, if not removed, cause genomic instability. Consequently, all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids (RDHs). Escherichia coli lacking RNase HI (rnhA) and RNase HII (rnhB) enzymes, the ΔrnhA ΔrnhB double mutant, accumulates RDHs in its DNA. These RDHs can convert into RNA-containing DNA lesions (R-lesions) of unclear nature that compromise genomic stability. The ΔrnhAB double mutant has severe phenotypes, like growth inhibition, replication stress, sensitivity to ultraviolet radiation, SOS induction, increased chromosomal fragmentation, and defects in nucleoid organization. In this study, we found that RNase HI deficiencyalso alters wild-type levels of DNA supercoiling. Despite these severe chromosomal complications, ΔrnhAB double mutant survives, suggesting that dedicated pathways operate to avoid or repair R-lesions. To identify these pathways, we systematically searched for mutants synthetic lethal (colethal) with the rnhAB defect using an unbiased color screen and a candidate gene approach. We identifiedboth novel and previously reported rnhAB-colethal and -coinhibited mutants, characterized them, and sorted them into avoidance or repair pathways. These mutants operate in various parts of nucleic acid metabolism, including replication fork progression, R-loop prevention and removal, nucleoid organization, tRNA modification,recombinational repair, and chromosome-dimer resolution, demonstrating the pleiotropic nature of RNase H deficiency.