Junction ribonuclease activity specified in RNases HII/2

Junction ribonuclease (JRNase) recognizes the transition from RNA to DNA of an RNA–DNA/DNA hybrid, such as an Okazaki fragment, and cleaves it, leaving a mono‐ribonucleotide at the 5′ terminus of the RNA–DNA junction. Although this JRNase activity was originally reported in calf RNase H2, some other RNases H have recently been suggested to possess it. This paper shows that these enzymes can also cleave an RNA–DNA/RNA heteroduplex in a manner similar to the RNA–DNA/DNA substrate. The cleavage site of the RNA–DNA/RNA substrate corresponds to the RNA/RNA duplex region, indicating that the cleavage activity cannot be categorized as RNase H activity, which specifically cleaves an RNA strand of an RNA/DNA hybrid. Examination of several RNases H with respect to JRNase activity suggested that the activity is only found in RNase HII orthologs. Therefore, RNases HIII, which are RNase HII paralogs, are distinguished from RNases HII by the absence of JRNase activity. Whether a substrate can be targeted by JRNase activity would depend only on whether or not an RNA–DNA junction consisting of one ribonucleotide and one deoxyribonucleotide is included in the duplex. In addition, although the activity has been reported not to occur on completely single‐stranded RNA–DNA, it can recognize a single‐stranded RNA–DNA junction if a double‐stranded region is located adjacent to the junction.