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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA replication frequently incorporates ribonucleoside monophosphates (rNMPs) instead of deoxyribonucleoside monophosphates (dNMPs).
• The presence of rNMPs in genomic DNA raises concerns about potential DNA damage and mismatches.
• RNase HII enzymes are crucial for removing rNMPs from DNA.

Purpose of the Study:

• To investigate the activity of Bacillus subtilis and Escherichia coli RNase HII enzymes.
• To determine the enzymes' efficacy in processing canonical, mismatched, and damaged rNMPs.
• To compare the repair mechanisms between bacterial and eukaryotic RNase HII orthologs.

Main Methods:

• Testing the endoribonuclease activity of E. coli and B. subtilis RNase HII.
• Utilizing canonical, mismatched, and damaged rNMPs as substrates.
• Analyzing the incision frequencies of various rNMP variants in DNA.

Main Results:

• E. coli RNase HII efficiently incised most rNMP variants, except for oxidized rNMPs where activity was reduced.
• B. subtilis RNase HII efficiently processed rAMP, rCMP, and rUMP but struggled with rGMP, even in canonical and mismatched pairs.
• B. subtilis RNase HII showed refractoriness to processing abasic and oxidized ribonucleotide lesions.

Conclusions:

• Bacterial RNase HII enzymes exhibit distinct intrinsic endoribonuclease activities for repairing rNMP errors.
• Not all rNMP incorporation errors are efficiently resolved by bacterial RNase HII.
• The recalcitrance of B. subtilis RNase HII to repair damaged rNMPs suggests the involvement of other repair pathways, similar to eukaryotic orthologs.