ARTICLE Received 27 Feb 2013 | Accepted 9 Jul 2013 | Published 5 Aug 2013 DOI: 10.1038/ncomms3273 OPEN Human endonuclease V is a ribonuclease specific for inosine-containing RNA Yoko Morita1, Toshihiro Shibutani1, Nozomi Nakanishi1, Kazuko Nishikura2, Shigenori Iwai1 & Isao Kuraoka1 Deamination of DNA bases can create missense mutations predisposing humans to cancer and also interfere with other basic molecular genetic processes; this deamination generates deoxyinosine from deoxyadenosine. In Escherichia coli, the highly conserved endonuclease V is involved in alternative excision repair that removes deoxyinosine from DNA. However, its exact activities and roles in humans are unknown. Here we characterize the FLJ35220 protein, the human homologue of E. coli endonuclease V, hEndoV as a ribonuclease specific for inosine-containing RNA. hEndoV preferentially binds to RNA and efficiently hydrolyses the second phosphodiester bond located 30 to the inosine in unpaired inosine-containing ssRNA regions in dsRNA. It localizes to the cytoplasm in cells. The ribonuclease activity is promoted by Tudor staphylococcal nuclease and detected on inosine-containing dsRNA created by the action of adenosine deaminases acting on RNA. These results demonstrate that hEndoV controls the fate of inosine-containing RNA in humans. 1 Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan. 2 The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. Correspondence and requests for materials should be addressed to I.K. (email: [email protected]). NATURE COMMUNICATIONS | 4:2273 | DOI: 10.1038/ncomms3273 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3273 lthough genomic DNA contains genetic information that to have a similar role8,15. However, the AER pathway has not yet should be error free to facilitate the proper functioning of been elucidated in mammalian cells, and substantial discrepancies Athe cell, DNA is prone to deterioration and modifications have been reported in the biochemical results of mammalian due to environmental and endogenous damage. Under physio- EndoV. Purified hEndoV from E. coli recognizes and cleaves logical conditions, deamination of DNA bases can occur both dsDNA and ssDNA containing deoxyinosine, by using spontaneously and at a physiologically significant rate via a nanomolar concentrations of the fluorescent substrate containing hydrolytic reaction. Three of the four DNA bases (cytosine, deoxyinosine15. Specific activity has been observed for the adenine and guanine) have amino groups. Deamination of these mEndoV in ssDNA containing deoxyinosine, but this activity is three bases generates the base analogues uracil, hypoxanthine and very poor in dsDNA containing deoxyinosine8. On the other xanthine, respectively1. Bases in DNA and nucleotides can be hand, Fladeby et al.16 showed that hEndoV neither cleaved nor deaminated both chemically and enzymatically. Deamination of bound to dsDNA substrates containing deoxyinosine under the DNA precursors can lead to the accumulation of noncanonical same conditions in which eEndoV cleaved efficiently. Moreover, nucleoside triphosphates in the cell’s nucleotide pool2,3. These mEndoV cleaves ssDNA substrates but has reduced activity for noncanonical nucleotides, such as dITP, dXTP and dUTP, can be dsDNA substrates8. incorporated into newly synthesized DNA, albeit less efficiently Here we purified recombinant hEndoV and characterized than regular DNA precursors. As deaminated bases in DNA have the function of this deoxyinosine 30 endonuclease. Our findings pairing specificities different from the original bases, these lesions indicate that hEndoV prefers RNA substrates over DNA have high mutagenic potential and may contribute to the substrates. Deoxyinosine is found in DNA, while inosine is incidence of genetic diseases and cancer. Because of these risks, found in RNA due to spontaneous or nitrosative deamination cells possess excision repair functions to prevent mutagenesis4. reactions and the incorporation of inosine triphosphate (ITP) Excision repair systems are highly conserved from bacteria to during transcription2,3. Inosine has been implicated in RNA humans4. There are several major DNA repair pathways, such as editing17–20, a post-transcriptional modification that alters the nucleotide excision repair (NER), which operates primarily on sequence of RNA from the original sequence encoded in the bulky helix-distorting damage caused by ultraviolet irradiation or DNA. Interestingly, RNA from brain tissue is the most frequently genotoxic chemicals, and base excision repair (BER), which is edited21. Thus, as hEndoV can cleave inosine-containing RNA (i- used for nonbulky and non–helix-distorting DNA modifications RNA) at specific sites, this RNA-editing activity may be a key induced by alkylation, oxidation and deamination. Deoxyuridine factor determining the fate of i-RNA. is a nucleoside formed when uracil is attached to a deoxyribose ring; this deamination product is subjected to BER, which is Results initiated by a uracil-DNA glycosylase that searches the genome to hEndoV amino-acid sequence. Five representative EndoV homo- locate the sites of damage and then catalyses the hydrolysis of the logues were aligned to examine similarities between bacteria N-glycosidic bond to release the lesion and generate an apurinic/ and mammals (Supplementary Fig. S1). A complementary DNA apyrimidinic (AP) site. Downstream enzymes subsequently pro- (cDNA) sequence for FLJ35220 (hEndoV), exhibiting homology cess this AP site and use the information from the un- 5 to eEndoV, was cloned from total human dermal fibroblast RNA. damaged strand to restore the original coding sequence . The encoded open reading frame predicted a product of 282 Deoxyinosine is a nucleoside formed when hypoxanthine amino-acid residues, with a calculated molecular mass of 31 kDa. is attached to a deoxyribose ring. Although deoxyinosine is Alignment of the amino-acid sequence with that of eEndoV repaired by alkyladenine DNA glycosylase (AAG) in BER, it revealed the following characteristics for hEndoV and eEndoV: is also subjected to alternative excision repair (AER), which is 6–8 identities ¼ 74/199 (37%), positives ¼ 108/199 (54%) and biochemically distinct from NER and BER . While NER gaps ¼ 19/199 (10%). The predicted active site in hEndoV involves the dual excision of the damaged flanking sites and (Asp52, Tyr91 or Glu100) was conserved from E. coli to humans, BER involves the cleavage of the N-glycosidic bond between the corresponding to Asp43, Tyr80 or Glu89 in eEndoV, that is, damaged base and the sugar in DNA, this AER pathway involves sites that had a significant effect on catalysis in Thermotoga the initiation of a single nick, strictly on one side of the base maritima22,23. damage in the DNA strand. The AER that has been most extensively defined by studies in E. coli involves a specific endonuclease called endonuclease V (E. coli endonuclease V: Properties of hEndoV. To examine the biological activities of the eEndoV), a product of the nfi gene in E. coli9. Studies on the protein encoded by the hEndoV cDNA, we prepared recombinant mutant strain have revealed a high frequency of A:T to G:C hEndoV with a glutathione S-transferase (GST) N-terminal tag in transitions following exposure to nitrous acid, which induces E. coli and purified the recombinant protein (Fig. 1a). As expected deoxyinosine in DNA10,11. Deoxyinosine has strong miscoding from the calculated relative molecular mass, purified hEndoV properties because it pairs with C. Purified eEndoV is a bearing the GST-tag migrated as a single band of approximately deoxyinosine 30 endonuclease that recognizes both double- 55 kDa in SDS–polyacrylamide gel electrophoresis analysis stranded and single-stranded DNAs (dsDNA and ssDNA, (Fig. 1a). Mutant proteins bearing alanine substitutions at Asp52, respectively) containing deoxyinosine and cleaves the second Tyr91 or Glu100 in hEndoV were purified as a single band by the and third phosphodiester bonds 30 to the mismatch of same procedures (Fig. 1b). deoxyinosine, leaving a nick with 30 hydroxyl and 50 phosphate To study the ability of hEndoV to cleave the second and third groups. Moreover, this enzyme has subsequently been shown to phosphodiester bonds 30 to the deoxyinosine, which is known have a broad substrate spectrum and acts at AP sites, urea as deoxyinosine 30 endonuclease activity in eEndoV, a 30-mer residues, base mismatches, flap DNA, pseudo Y structures and ssDNA oligonucleotide was synthesized containing deoxyinosine loops, and hairpins6,12,13. As it cannot release deoxyinosine or the residue 14 (Fig. 1c). hEndoV, like eEndoV, could cleave ssDNA damaged bases, eEndoV is thought to require another enzyme to containing a deoxyinosine but was inefficient at cleaving ssDNA completely repair the DNA containing deoxyinosine8,14.InE. coli containing deoxyadenosine or deoxyuridine, which are cytosine and some other bacteria, there is an eEndoV-dependent AER deamination products (Fig. 1c). However, although hEndoV pathway for the removal of inosine from DNA. In mammalian could cleave ssDNA containing deoxyinosine, its activity was low cells, hEndoV and mouse EndoV (mEndoV) have been proposed (see Fig. 1c, lanes 2 and 5). All mutations, that is, D52A, Y91A 2 NATURE COMMUNICATIONS | 4:2273 | DOI: 10.1038/ncomms3273 | www.nature.com/naturecommunications & 2013