RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome wide.

RNA:DNA hybrids compromise replication fork progression and genome integrity in all cells. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate strongly in noncoding RNAs and 5'-UTRs of coding sequences. For Δ rnhB , hybrids accumulate preferentially in untranslated regions and early in coding sequences. We show that hybrid accumulation is particularly sensitive to gene expression in Δ rnhC cells. DNA replication in Δ rnhC cells is disrupted, leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts cause mutagenesis and shape genome organization.