# Bacillus subtilis RNase HII Is Inefficient at Processing Guanosine Monophosphate and Damaged Ribonucleotides

## TL;DR

This study shows that Bacillus subtilis RNase HII can correct some ribonucleotide errors in DNA but is inefficient at processing damaged or mismatched guanosine monophosphates.

## Contribution

The study reveals novel differences in RNase HII activity between bacterial species and identifies limitations in processing damaged ribonucleotides.

## Key findings

- Bacillus subtilis RNase HII efficiently incises rAMP, rCMP, and rUMP but is inefficient with rGMP.
- Bacillus subtilis RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions.
- The inefficiency of Bacillus subtilis RNase HII in repairing damaged rNMPs resembles eukaryotic RNase H2 orthologs.

## Abstract

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.

Bacillus subtilis
RNase HII corrects ribonucleotide errors and mismatched ribonucleotides, but not damaged ribonucleotides. Mismatched ribonucleotides would also serve as substrates for mismatch repair while damaged ribonucleotides would be addressed through base excision repair.