<scp> <i>Bacillus subtilis</i> RNase HII </scp> Is Inefficient at Processing Guanosine Monophosphate and Damaged Ribonucleotides

During one round of DNA replication, nearly 2000 ribonucleoside monophosphates (rNMPs) are incorporated in place of their cognate deoxyribonucleoside monophosphates (dNMPs). Given their high rate of insertion, genomic DNA could contain rNMPs that are damaged or mismatched. Here, we test the activity of Bacillus subtilis and Escherichia coli RNase HII on canonical, mismatched, and damaged rNMPs. We show that E. coli RNase HII is adept at incising most rNMP variants from DNA at similar frequencies, with the exception of an oxidized rNMP, where endoribonuclease activity is sharply reduced. In contrast, B. subtilis RNase HII efficiently incises rAMP, rCMP, and rUMP but is inefficient at processing rGMP in both a canonical and mismatched base pair. We test damaged ribonucleotides and find that B. subtilis RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions. Our work shows that bacterial RNase HII enzymes have different intrinsic endoribonuclease activity toward the repair of canonical, mismatched, and damaged rNMPs, demonstrating that not all rNMP errors provoke efficient resolution. Our finding that B. subtilis RNase HII is recalcitrant to repairing damaged rNMPs resembles what is observed for eukaryotic RNase H2 orthologs, suggesting that other repair processes are necessary to resolve damaged rNMPs.