MCDB Dissertation Defense Seminar> Bacterial Ribonuclease HII and Ribonucleotide Excision Repair

Although RNA-DNA hybrids are essential to the perpetuation of life, if left unresolved, ribonucleoside monophosphates (rNMPs) embedded in the genome are a threat to faithful DNA replication, genomic integrity, and genetic inheritance. Single rNMPs, which differ from deoxyribonucleoside monophosphates (dNMPs) by a single 2′ –OH group, often become misincorporated in the genome by replicative DNA polymerases during DNA replication. Although DNA polymerases have a steric exclusion mechanism to prevent rNTPs from accessing their active site, rNTPs outnumber dNTPs in the cell and as a result are still frequently used as substrates during synthesis. rNMPs, also known as “sugar errors,” that go unrepaired can lead to increased mutagenesis in the form of transitions, single-stranded breaks, and even lethal double-stranded breaks (DSBs) due to the reactivity of the 2′ –OH. However, cells have evolved to combat sugar errors through ribonucleotide excision repair (RER), which is initiated by Ribonuclease HII/H2 (RNase HII). RNase HII is a Type 2 RNase H enzyme that makes a 5′ incision to a rNMP in double-stranded DNA (dsDNA), permitting subsequent entry by a DNA polymerase. The polymerase extends from the 3′ –OH of the nick, displacing the downstream DNA containing the rNMP to generate a 5′ flap, which can be resolved by a flap endonuclease (FEN). Finally, DNA ligase seals the gap in the backbone. The junction-sensing module of RNase HII is a critical structural element that gives the protein its unique ability to specifically recognize single rNMPs by their 2′ –OH. RER has been previously studied in eukaryotes, archaea, and bacteria, although the breadth of lesions addressed by bacterial RNase HII enzymes has not yet been established. We performed in vitro assays using RNase HII purified from Escherichia coli and Bacillus subtilis and dsDNA substrates containing single canonical, mismatched, and damaged rNMPs. Specifically, E. coli RNase HII (EcoRNase HII) was equally active on all four canonical rNMPs, including rUMP, while B. subtilis RNase HII (BsuRNase HII) was inefficient at processing rGMP. In a mismatched context, EcoRNase HII activity on rAMP and rGMP was unchanged from that on canonical rNMPs. Conversely, BsuRNase HII activity on rG:dT was significantly reduced from that on rG:dC. Further, EcoRNase HII demonstrated weak activity on r8oG:dC, while BsuRNase HII could not resolve r8oG:dC nor rOH:dC. This observation is similar to that reported for archaeal and eukaryotic RNase HII homologs. Together, our results show that bacterial RNase HII proteins have different substrate preferences. Surprisingly, both EcoRNase HII and BsuRNase HII are able to incise rUMP, suggesting that RER, and not base excision repair (BER), is the primary pathway for rUMP repair. Moreover, mismatched rNMP errors appear to be substrates for both RER and mismatch repair (MMR). Finally, damaged rNMP errors in B. subtilis must be repaired by an alternative pathway, potentially BER, although this has not yet been tested.