title: Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA-DNA junction to the RNA/DNA heteroduplex.
abstract: 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.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan. ohtani@ttck.keio.ac.jp
authors.forename: Naoto
authors.initials: N
authors.lastname: Ohtani
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Itaya
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: C022262:Okazaki fragments; D012313:RNA; D004247:DNA; C091139:ribonuclease HII; D016914:Ribonuclease H