Junction ribonuclease: A ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA-DNA junction to the RNA/DNA heteroduplex

Naoto Ohtani, Masaru Tomita, Mitsuhiro Itaya

Research output: Contribution to journalArticle

15 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)517-526
Number of pages10
JournalBiochemical Journal
Volume412
Issue number3
DOIs
Publication statusPublished - 2008 Jun 15

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Keywords

  • Junction ribonuclease (JRNase)
  • Okazaki fragment
  • RNA-DNA junction
  • Ribonuclease H (RNase H)
  • Ribonuclease HII (RNase HII)
  • Thermus thermophilus HB8

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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