Cleavage of a DNA–RNA–DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA–RNA junction with prokaryotic RNases HII

Abstract

We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA–RNA–DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5′-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA–RNA junction (5′-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA.