The human homolog of Escherichia coli endonuclease V is a nucleolar protein with affinity for branched DNA structures.

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 3' 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 3'-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

Human ENDOV binds branched DNA substrates.

<p>The affinities of <i>E. coli</i> and human endonuclease V for different DNA substrates were tested by electrophoretic mobility shift assay. Substrates used were: (<b>A</b>): Undamaged DNA, (<b>B</b>): Hypoxanthine, (<b>C</b>): 5′-flap, (<b>D)</b>: 3′-flap, (<b>E</b>): pseudo-Y, (<b>F</b>): fork, (<b>G</b>): 3-way junction and (<b>H</b>): Holliday junction (asterisk indicates the ³²P-labelled strands). 2–8 pmol <i>E. coli</i> endoV and 1.6–6.4 pmol ENDOV enzymes were assayed with 10 fmol substrates as indicated. All experiments were repeated 2 to 3 times and a representative assay is shown. Bound substrates relative to free were quantified and are shown to the right. F = free DNA, B = bound DNA, - = no enzyme added, filled circles = <i>E. coli</i> endoV; open circles = ENDOV.</p

The EGFP-ENDOV fusion protein localises to nucleoli and cytoplasm.

<p>Confocal imaging of HeLa cells overexpressing GFP-ENDOV (<b>A</b>) probed with an anti-fibrillarin antibody (<b>B</b>), an overlay of the two (<b>C</b>) or GFP only (<b>D</b>).</p

The RK ENDOV mutant has lost its affinity for branched DNA substrates.

<p>Three ENDOV site specific mutants, RK (R248E/K249E double mutant), Y91A, and Wg (residues P90–S93 replaced with 4 glycins), wild type enzyme (3.2 and 6.4 pmol) and <i>E. coli</i> endoV (4 pmol) were tested for their ability to bind branched DNA substrates. Substrates tested were: (<b>A</b>): 5′-flap, (<b>B</b>): 3′-flap, (<b>C</b>): pseudo-Y, (<b>D</b>), fork, (<b>E</b>): 3-way junction and (<b>F</b>) Holliday junction. F = free DNA, B = bound DNA, - = no enzyme added.</p

Location of structural elements in endonuclease V.

<p>(<b>A</b>) Structure of <i>Tma</i> endoV binding to deaminated DNA (PDB code 2W35 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047466#pone.0047466-Dalhus1" target="_blank">[23]</a>) showing the central location of the strand-separating wedge (PYIP motif) close to the damage recognition pocket. (<b>B</b>). Homology model of human ENDOV showing the location of mutated residues Arg248 and Lys249 (yellow), as well as residues forming the PYVS motif in exon 3 (yellow), which were also mutated. A loop comprising residues 164–180 could not be reliably modelled and is not included (dashed line).</p

Schematic representation of splicing of human, other tetrapod and sea urchin <i>ENDOV</i> mRNAs.

<p>(<b>A</b>) The segments of the <i>ENDOV</i> transcripts encoding the conserved protein core are spliced identically, including the intron phases, in rodents, pig, chicken, frog and the echinoderm sea urchin. The position of the start (ATG) and stop (asterisk) codons are indicated above the relevant exons (not shown to scale), while intron phases are shown above the introns. These are defined as the position of the intron within a codon, with phase 0, 1, and 2 placed before the first nucleotide, after the first nucleotide, and after the second nucleotide, respectively. The structurally disordered and poorly conserved C-terminus of ENDOV is encoded by one or more 3′ exons and has variable length, <i>e.g.</i> ∼85 and ∼5 residues in mouse and frog, respectively. (<b>B</b>) Most previously published spliced human mRNAs are lacking one or several 5′ exons, in particular exon 3, while alternative splicing at the 3′ end results in multiple variants. Four representative full length mRNAs are shown with accession numbers from GenBank <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047466#pone.0047466-Benson1" target="_blank">[29]</a>.</p

Upregulation of <i>ENDOV</i> transcription during quiescence.

<p>(<b>A</b>) Human embryonic fibroblasts were arrested in G0 by serum starvation at confluence and released by replating 1∶4 in culture medium with serum. <i>ENDOV</i> transcript levels (exons 2 to 3) were measured during cell cycle progression after G0 release at indicated time points by qRT-PCR. G0 cells were used as the reference for calculations. The average of 3 parallels (same RNA) was calculated and standard deviation is shown. Cell cycle distribution was monitored using propidium iodide staining followed by flow cytometry after release from the block. The percentage of cells in each cell cycle is presented in the table. The experiment was repeated twice with similar results. (<b>B</b>) Nothern blot analysis of <i>ENDOV</i> mRNA. mRNA was isolated from human fibroblasts that were unsynchronised (u), G0 arrested (G0) and allowed to proliferate for 24 hours (t₂₄), separated by electrophoresis and transferred to a nylon membrane. Hybridisation signals with probes spanning exons 4–8, exon 10, exon 3 of <i>ENDOV</i> and for β<i>-ACTIN</i> are shown. M is the RNA size standard as indicated (in kilobases).</p

Expression of <i>ENDOV</i> in human cancers.

<p>Transcript profiling was performed using a cancer tissue qPCR array and primers amplifying exons 6 to 8 of <i>ENDOV</i>. cDNA levels are normalised to β<i>-ACTIN</i>. <i>ENDOV</i> mRNA levels were calculated relative to the tissue with lowest expression level (adrenal gland). Tumour and normal tissue were grouped according to their origin and the average and standard deviation were calculated for each group. The experiment was performed twice with similar results.</p