Val143 of human ribonuclease H2 is not critical for, but plays a role in determining catalytic activity and substrate specificity.

Ribonuclease H2 (RNase H2) exhibits both single ribonucleotide excision activity (activity A) and RNA strand degrading activity (activity B). Val143 of human RNase H2 is located at the active site and is conserved in eukaryotic RNase H2. In this study, we explored the role of Val143 in catalytic activity and substrate specificity. Nineteen single variants at amino acid position 143 were expressed in E. coli, and all variants except for V143C and V143M were purified from the cells. When the activity of the wild-type human RNase H2 (WT) was set as 100%, the relative activities A and B of the 17 variants were in the range of 0.05-130 and 0.02-42%, respectively. When the ratio of the relative activity A to the relative activity B of WT was set as 1, the ratios of the 17 variants were in the range of 0.2-5.7. This indicates that valine is optimal for balancing the two activities. The ratios for V143Y and V143W were relatively high (5.6 and 5.5, respectively), suggesting that the bulky residues like tyrosine and tryptophan at position 143 caused steric hindrance with the 2'-OH of the sugar moiety of the ribonucleotide at the 5' side of the scissile phosphodiester bond. The ratio for V143Q was relatively low (0.2). These results suggested that Val143 is not critical for, but plays a role in determining catalytic activity and substrate specificity.

Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations.

Mammalian RNase H2 is a heterotrimeric enzyme consisting of one catalytic subunit (A) and two accessory subunits (B and C). RNase H2 is involved in the removal of a single ribonucleotide embedded in genomic DNA and removal of RNA of RNA/DNA hybrids. In humans, mutation of the RNase H2 gene causes a severe neuroinflammatory disorder Aicardi-Goutières syndrome (AGS). Here, we examined the activity and stability of six recombinant human RNase H2 variants bearing one AGS-causing mutation, A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G, or C-R69W. The activity of A-G37S was 0.3-1% of that of the wild-type RNase H2 (WT), while those of other five variants were 51-120%. In circular dichroism measurement, the melting temperatures of variants were 50-53°C, lower than that of WT (56°C). These results suggested that A-G37S had decreased activity and stability than WT, while other five variants had decreased stability but retained activity. In gel filtration chromatography of the purified enzyme preparation, WT migrated as a heterotrimer, while A-R291H eluted in two separate peaks containing either the heterotrimer or only the A subunit, suggesting that some AGS-causing mutations affect the heterotrimer-forming stability of RNase H2.

RNA/DNA structures recognized by RNase H2.

Ribonuclease H (RNase H) [EC 3.1.26.4] is an enzyme that specifically degrades RNA from RNA/DNA hybrids. Since its discovery in 1969, the enzyme has been extensively studied for its catalytic mechanism and physiological role. RNase H has been classified into two major families, Type 1 and Type 2. Type 1 enzymes are designated RNase HI in prokaryotes and RNase H1 in eukaryotes, while Type 2 enzymes are designated RNase HII in prokaryotes and RNase H2 in eukaryotes. Type 2 enzymes are able to cleave the 5'-phosphodiester bond of one ribonucleotide embedded in a DNA double strand. Recent studies have shown that RNase H2 is involved in excision of a single ribonucleotide embedded in genomic DNA and removal of an R-loop formed in cells. It is also involved in double-strand break of DNA and its repair. In this review, we aim to outline the structures recognized by RNase H2.