Comparison of biochemical properties of RNases HII from E. coli, Geobacillus stearothermophilus and Thermus thermophilus

Abstract

RNases H (EC 3.1.26.4) are a family of enzymes participating in removal of ribonucleotides from double-stranded DNA by hydrolyzing their phosphodiester bonds. Despite a long history of research, several important aspects of RNase HII functioning are poorly known including optimal pH, salts and thermal stability. This lack of empirical data hampers the selection of an optimal RNase HII for a specific practical application. In the present study, we compared biochemical properties of previously cloned RNases HII from E. coli, Geobacillus stearothermophilus and Thermus thermophilus: optimal temperature, pH, salts, divalent cofactors, thermal stability and specific activity. As expected, the most thermostable enzyme was Tth RNase HII, and Mg2+ was the most preferential cofactor for all RNases. Gst RNase HII was partially inhibited by K+ ions, while other enzymes did not demonstrate preferences for any salt. The enzymes from E. coli and G. stearothermophilus were typical RNases HII, while Tth RNase HII was a JRNase (junction ribonuclease). All three RNases did not cleave a DNA-RNA3-DNA2-RNA1-DNA/DNA substrate. The presented results will facilitate usage of RNases HII in practical applications and provide a basis for further comparative studies of RNases HII from various organisms.