Substrate specificity for bacterial RNase HII and HIII is influenced by metal availability.

We tested the activity of four predicated RNase H enzymes including two RNase HI-type enzymes in addition to RNase HII (RnhB) and RNase HIII (RnhC) on several RNA:DNA hybrid substrates with different divalent metal cations. We found that the two RNase HI-type enzymes YpdQ and YpeP failed to show activity on the three substrates tested. RNase HII and RNase HIII cleaved all substrates tested although activity was dependent on the metal made available. We show that B. subtilis RNase HII and RNase HIII are both able to incise 5' to a single rNMP. We show that RNase HIII incision at a single rNMP occurs most efficiently with Mn2+ , an activity we found to be conserved among other Gram-positive RNase HIII enzymes. Characterization of RNase HII and HIII with metal concentrations in the physiological range showed that RNase HII can cleave at single rNMPs embedded in DNA while RNase HIII is far less effective. Further, using metal concentrations within physiological range, RNase HIII efficiently cleaved longer RNA:DNA hybrids lacking an RNA:DNA junction while RNase HII is much less effective. Phenotypic analysis shows that cells with an rnhC deletion are sensitive to hydroxyurea (HU). In contrast, cells with an rnhB deletion show wild type growth in the presence of HU supporting the hypothesis that RNase HII and HIII have distinct substrate specificities in vivo This work demonstrates how metal availability influences substrate recognition and activity of RNase HII and HIII providing insight into their function in vivo IMPORTANCE Ribonuclease H (RNase H) represents a class of proteins that cleave RNA:DNA hybrids helping resolve R-loops and Okazaki fragments as well as initiating the process of ribonucleotide excision repair (RER). We investigated the activity of four Bacillus subtilis RNase H enzymes finding that only RNase HII and HIII have activity and that their substrate preference is dependent on metal availability. To understand factors that contribute to RNase HII and RNase HIII substrate preference, we show that in the presence of metal concentrations within physiological range, RNase HII and HIII have distinct activities on different RNA:DNA hybrids. This work provides insight into how RNase HII and HIII repair the broad range of RNA:DNA hybrids that form in Gram-positive bacteria.