The genome of an extremely thermophilic bacterium, Thermus thermophilus HB8, contains a single ORF (open reading frame) encoding an RNase-HII-like sequence. Despite the presence of significant amino acid sequence identities with RNase (ribonuclease) HII enzymes, the ORF TTHA0198 could not suppress the temperature-sensitive growth defect of an RNase-H-deficient Escherichia coli mutant and the purified recombinant protein could not cleave an RNA strand of an RNA/DNA heteroduplex, suggesting that the TTHA0198 exhibited no RNase H activity both in vivo and in vitro. When oligomeric RNA–DNA/DNAs were used as a mimic substrate for Okazaki fragments, however, the protein cleaved them only at the 5′ side of the last ribonucleotide at the RNA–DNA junction. In fact, the TTHA0198 protein prefers the RNA–DNA junction to the RNA/DNA hybrid. We have referred to this activity as JRNase (junction RNase) activity, which recognizes an RNA–DNA junction of the RNA–DNA/DNA heteroduplex and cleaves it leaving a mono-ribonucleotide at the 5′ terminus of the RNA–DNA junction. E. coli and Deinococcus radiodurans RNases HII also cleaved the RNA–DNA/DNA substrates at the same site with a different metal-ion preference from that for RNase H activity, implying that the enzymes have JRNase activity as well as RNase H activity. The specialization in the JRNase activity of the RNase HII orthologue from T. thermophilus HB8 (Tth-JRNase) suggests that the JRNase activity of RNase HII enzymes might be independent of the RNase H activity.
Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8.
