Characterization of RNase HII substrate recognition using RNase HII-argonaute chimaeric enzymes from Pyrococcus furiosus

Sayaka Kitamura, Kosuke Fujishima, Asako Sato, Daisuke Tsuchiya, Masaru Tomita, Akio Kanai

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA-DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA-DNA hybrids than for RNA-RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA-RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth β-sheet and the fifth α-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII-Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe 114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes.

Original languageEnglish
Pages (from-to)337-344
Number of pages8
JournalBiochemical Journal
Volume426
Issue number3
DOIs
Publication statusPublished - 2010 Mar 15

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Keywords

  • Archaeon
  • Argonaute
  • Double-stranded RNA (dsRNA)
  • RNA-DNA duplex
  • Ribonuclease H
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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