The Human Homolog of Escherichia coli Endonuclease V Is a Nucleolar Protein with Affinity for Branched DNA Structures

et al. (2012) The Human Homolog of Escherichia coli Endonuclease V Is a Nucleolar Protein with Affinity for Branched DNA Structures. PLoS ONE 7(11): e47466. doi:10.1371/journal.pone.0047466 The Human Homolog of Escherichia coli Endonuclease V Is a Nucleolar Protein with Affinity for Branched DNA Structures Cathrine Fladeby 0 Erik Sebastian Vik 0 Jon K. Laerdahl 0 Christine Gran Neurauter 0 Julie E. Heggelund 0 Eirik Thorgaard 0 Pernille Strm-Andersen 0 Magnar Bjra s 0 Bjrn Dalhus 0 Ingrun Alseth 0 Zhongjun Zhou, The University of Hong Kong, Hong Kong 0 1 Department of Microbiology, Oslo University Hospital HF and University of Oslo , Rikshospitalet, Oslo , Norway , 2 Department of Medical Biochemistry, Oslo University Hospital HF and University of Oslo , Rikshospitalet, Oslo , Norway , 3 Centre for Molecular Biology and Neuroscience (CMBN), Oslo University Hospital HF and University of Oslo , Rikshospitalet, Oslo , Norway Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx) bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV), encoded by the nfi gene, which cleaves the second phosphodiester bond 39 of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV), many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 39-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription. - Funding: The study was supported by The Norwegian Research Council and The Norwegian Cancer Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The genomes of all organisms are constantly challenged by agents, produced inside the cell or in the environment, that cause damage to the DNA. DNA base damage may lead to errors in replication and transcription, compromising the integrity of the genome. Three of the four bases present in DNA (cytosine, adenine, and guanine) contain an exocyclic amino group. Loss of this group by deamination occurs spontaneously under physiological conditions via a hydrolytic reaction [1,2]. This process is greatly enhanced by agents such as reactive oxygen radicals, UV radiation, heat, ionizing radiation, nitrous acid, nitric oxide, and sodium bisulfite [37]. It is estimated that a few hundred amino groups are lost from the DNA bases spontaneously in each cell every day, most frequently from cytosine bases. Adenine deamination occurs only at a rate of 23% compared to that of cytosine [8]. Deamination of cytosine and adenine produces uracil and hypoxanthine (Hx), respectively, both having miscoding properties. In addition, Hx in DNA might be the result of misincorporation of 29-deoxyinosine triphosphate (dITP) during DNA replication [9]. In this case dITP is incorporated opposite cytosine and is also read as guanine by the DNA polymerases. Thus, at least in Escherichia coli, dITP incorporation is nonmutagenic [10]. Whereas uracil in DNA is removed by uracil DNA glycosylases [11], the principal enzyme for removal of Hx in E. coli is endonuclease five (endoV) encoded by the nfi gene [12]. This enzyme binds to and cleaves the second phosphodiester bond 39 to Hx in an Mg2+ dependent manner generating 39-OH and 59-P termini [13,14]. Endonuclease V does not on its own remove the damage from DNA and additional proteins are thus required to complete repair. This process is poorly understood but has been shown to be reconstituted with recombinant endoV, DNA polymerase I and DNA ligase [15]. E. coli cells lacking endoV have a normal spontaneous mutation frequency, however upon exposure to nitrous acid nfi2 cells are mutators showing elevation in ATRGC and GCRAT transition as well as GCRCG transversion mutations [16]. E. coli endoV is a rather promiscuous enzyme acting on different substrates including uracil [17,18], xanthine (deaminated guanine) [19], apurinic/apyrimidinic (AP) sites [14], urea residues [14], mismatches [20] and also structure substrates such as insertion and deletion loops, 59-flaps, hairpins and pseudo-Y structures [21]. The ability of E. coli endoV to recognize all three deamination products in DNA is unique and is not shared by any of the other known repair enzymes. Finally, is has been shown that endoV from Thermotoga maritima (Tma) possesses both 59 and 39 exonuclease activities and a potential role for these activities in end-processing after Hx incision was suggested [22]. The 3-dimensional structure of Tma endoV in complex with Hx-containing DNA was recently determined [23]. The structure reveals the presence of a wedge motif (PYIP) involved in damage detection and DNA strand separation at the site of the lesion. The deaminated adenine lesion is rotated approximately 90u into a recognition pocket where it is tightly coordinated by hydrogen-bonding interactions. Homologs of endoV are widespread in nature and are found in all three domains of life [23]. In addition to E. coli, endoV homologs have been characterized from Archaeglobus fulgidus [24], T. maritima [25], Ferroplasma acidarmanus (in fusion with O6alkylguanine-DNA alkyltransferase active site domain) [26] and Salmonella typhimurium [27], however knowledge about the eukaryotic counterparts is sparse. cDNA for endoV from mice has been cloned, however no robust enzyme activity for Hx or other tested substrates were found [28]. In this work we have characterized the human variant of endonuclease V by identification of isoforms, subcellular localization and biochemical assays. Materials and Methods Ethics statement A commercially available tissue array was used and ethical principles maintained by the manufacturer (Origene) (http:// www.origene.com/Tissue/Tissue_QC.aspx). Bioinformatics analysis Protein and mRNA derived sequences were obtained from GenBank [29] and other NCBI database resources [30] and f (...truncated)