Structural basis for incision at deaminated adenines in DNA and RNA by endonuclease V

Progress in Biophysics and Molecular Biology 117 (2015) 134e142

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Review

Structural basis for incision at deaminated adenines in DNA and RNA by endonuclease V Bjørn Dalhus a, b, *, Ingrun Alseth b, Magnar Bjørås b a b

Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, PO Box 4950, Nydalen, N-0424 Oslo, Norway Department of Microbiology, Clinic for Diagnostics and Intervention, Oslo University Hospital, Rikshospitalet, PO Box 4950, Nydalen, N-0424 Oslo, Norway

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 28 March 2015

Deamination of the exocyclic amines in adenine, guanine and cytosine forms base lesions that may lead to mutations if not removed by DNA repair proteins. Prokaryotic endonuclease V (EndoV/Nfi) has long been known to incise DNA 30 to a variety of base lesions, including deaminated adenine, guanine and cytosine. Biochemical and genetic data implicate that EndoV is involved in repair of these deaminated bases. In contrast to DNA glycosylases that remove a series of modified/damaged bases in DNA by direct excision of the nucleobase, EndoV cleaves the DNA sugar phosphate backbone at the second phosphodiester 30 to the lesion without removing the deaminated base. Structural investigation of this unusual incision by EndoV has unravelled an enzyme with separate base lesion and active site pockets. A novel wedge motif was identified as a DNA strand-separation feature important for damage detection. Human EndoV appears inactive on DNA, but has been shown to incise various RNA substrates containing inosine. Inosine is the deamination product of adenosine and is frequently found in RNA. The structural basis for discrimination between DNA and RNA by human EndoV remains elusive. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Endonuclease V Deamination Hypoxantine Inosine

Contents 1. 2.

3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Endonuclease V e activity and role in DNA/RNA metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 2.1. Discovery, activities, mechanism and role of EndoV in prokaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 2.2. Mammalian EndoV e ribonuclease activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Endonuclease V e structures and recognition of substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 3.1. Prokaryotic EndoV in complex with deaminated DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 3.2. Prokaryotic EndoV in complex with loop DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 3.3. Structural basis for RNA incision by human EndoV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

1. Introduction Three of the four nucleobases in DNA can be deaminated at their primary amine positions, whereby the amino group is replaced by a

* Corresponding author. Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, PO Box 4950, Nydalen, N-0424 Oslo, Norway. E-mail address: [email protected] (B. Dalhus). http://dx.doi.org/10.1016/j.pbiomolbio.2015.03.005 0079-6107/© 2015 Elsevier Ltd. All rights reserved.

carbonyl moiety. Thus, the deaminated bases will have altered base pairing properties compared to the unmodified variants. Adenine is deaminated to form hypoxanthine, guanine to xanthine or oxanine, and finally cytosine is deaminated to form uracil (Fig. 1a). Deamination can be caused by spontaneous hydrolysis (Lindahl, 1993; Shen et al., 1994), by reaction with endogenous or exogenous factors, or by enzymatic reactions. For example, nitrate or nitrite metabolism, as well as chronic inflammation and macrophage

B. Dalhus et al. / Progress in Biophysics and Molecular Biology 117 (2015) 134e142

Fig. 1. Base deamination. (a) Deamination of adenine, guanine and cytosine produce hypoxanthine (Hx), xanthine (Xa) and oxanine (Ox), and uracil, respectively. (b) Hypoxanthine forms base pair with cytosine. (c) EndoV hydrolyses the second phosphodiester group 30 of the deaminated base (Hx).

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(Nguyen et al., 1992; Wink et al., 1991). Deamination of bases may also be catalysed by enzymes such as AID (activation-induced cytidine deaminase) in order to form uracil in DNA for antibody diversification (Muto et al., 2000; Revy et al., 2000; Muramatsu et al., 2000). All the deaminated bases in DNA may give rise to mutations due to misincorporation and formation of non-canonical basepairs during replication (Hill-Perkins et al., 1986; Wuenschell et al., 2003; Yasui et al., 2008). The deaminated DNA bases can cause mutations and cancer predispositions (Demple and Linn, 1982; Schouten and Weiss, 1999; Hussain et al., 2003; Nguyen et al., 1992; Wink et al., 1991). Particularly, for deaminated adenine, an A/T to G/C transition mutation may arise as hypoxanthine mispairs with cytosine (Fig. 1b) (Hill-Perkins et al., 1986; Schouten and Weiss, 1999). Related to this, members of the large APOBEC family, which convert cytosine to uracil in RNA or DNA, are believed to be the source for a large fraction of mutations found in tumors (Beale et al., 2004; Nik-Zainal et al., 2012; Roberts et al., 2013; Roberts and Gordenin, 2014). Another mechanism for introduction of deaminated bases in DNA is misincorporation of the nucleotides dUTP and dITP (deoxyinosine triphosphate; hypoxanthine is the base of deoxyinosine and inosine in DNA and RNA, respectively; Fig. 1a) by DNA polymerases (Budke and Kuzminov, 2006; Mathews, 2006; Pang et al., 2012). This misincorporation is mainly a non-mutagenic process since the DNA polymerases inserts adenine opposite uracil and cytosine opposite hypoxanthine. To avoid the mutagenic effect, the deaminated bases in DNA must be detected and removed. The most common deamination product, uracil, is removed via the Base Excision Repair (BER) pathway by way of uracil DNA glycosylases UNG and SMUG1 (Haushalter et al., 1999; Kavli et al., 2002; Olsen et al., 1989). Hypoxanthine and xanthine are also removed by BER glycosylases such as AlkA, Nei and Mug in Escherichia coli (Lee et al., 2010b; Saparbaev and Laval, 1994; Terato et al., 2002), and AAG in human cells (O'Brien and Ellenberger, 2004; Saparbaev and Laval, 1994). In addition to excision of hypoxanthine and xanthine bases by BER glycosylases, endonuclease V (EndoV/Nfi) has also been implicated in removal of these lesions in DNA. In contrast to DNA where deamination of bases is normally regarded a damage caused by spontaneous hydrolysis, nitrosative stress or misincorporation, bases in RNA are frequently edited, with the adenine-to-inosine modification being a frequent event (reviewed in e.g. (Bass, 2002; Gray, 2012)). In RNA, the enzymes ADAR (adenosine deaminase acting on RNA) and ADAT (adenosine deaminase acting on tRNA) deaminate adenosines in mRNA/noncoding RNAs and tRNAs, respectively, to form inosine as a way to generate transcriptome diversity (Keegan et al., 2004; Tsutsumi et al., 2007). The role of hypoxanthine/inosine in DNA and RNA has recently been discussed (Alseth et al., 2014). In short, within DNA, hypoxanthine is a premutagenic lesion, while in RNA, it is an essential, enzymatically generated modification introduced to give transcriptome variety. 2. Endonuclease V e activity and role in DNA/RNA metabolism 2.1. Discovery, activities, mechanism and role of EndoV in prokaryotes

activation produce reactive nitrogen and oxygen species (RNS/ROS) such as nitric oxide and superoxide (Dedon and Tannenbaum, 2004; Thomas et al., 2008). The RNS and ROS may further react with the exocyclic amines of DNA nucleobases. RNS have been proposed to cause cytotoxic and mutagenic DNA damage via direct reaction with DNA or via formation of DNA adduct-forming electrophiles from reactions with lipids, proteins and carbohydrates

The DNA incision activity of E. coli EndoV, encoded by the nfi gene, was first reported for DNA substrates containing uracil, apurinic/apyrimidinic sites, and various products formed by treatment of DNA with UV light, X-rays, acids or OsO4 (Demple and Linn, 1982; Gates and Linn, 1977a, 1977b). Later, in vitro studies revealed that E. coli EndoV incised a plethora of DNA substrates, including

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hypoxanthine, xanthine, urea (Yao et al., 1994), and base mismatches (Yao and Kow, 1994). E. coli EndoV is also capable of cleaving insertion/deletion mismatches, flap and pseudo Y structures, which are all characterized by a discontinuous/distorted DNA helix. Investigations of EndoV from the hyperthermophilic bacteria Thermotoga maritima (Tma) have revealed incision activity for similar substrates (Huang et al., 2001, 2002), and particularly, Tma EndoV can recognize and incise DNA at all deaminated bases (Feng et al., 2005). Both the E. coli and Tma EndoV have high affinity for the hypoxanthine substrate as well as the incised product (Huang et al., 2001; Yao and Kow, 1995). Recently, EndoV from the hyperthermophilic archea Pyrococcus furiosus was shown to incise at hypoxanthine both in DNA and RNA, an exhibited strong binding to the incised DNA product (Kiyonari et al., 2014). The E. coli and Tma EndoV hydrolyse the second phosphodiester bond 30 of a deaminated base using Mg2þ as a cofactor (Huang et al., 2001; Yao et al., 1994). This unique incision mechanism, with a onenucleotide off-set between the modified base and the point of incision (Fig. 1c) is not shared by other DNA repair proteins involved in single-base lesion recognition, and contrasts DNA glycosylases in the Base Excision Repair (BER) pathway, which remove damaged bases by hydrolytic cleavage of the N-glycosylic bond, leaving an abasic site for downstream processing (Karran et al., 1980; Lindahl, 1974; Seeberg et al., 1995). Since EndoV does not remove the lesion using an exonuclease activity, other factors are required to complete the repair cycle, but the details are yet to be established. Although the downstream processing of EndoV-mediated nicks remains elusive, a short repair patch spanning only 2e3 nucleotides to either side of a hypoxanthine has been suggested (Weiss, 2008), and a recent study reported on the in vitro reconstitution of a possible EndoV-mediated pathway for repair of hypoxanthine in DNA using recombinant EndoV, DNA polymerase I and DNA ligase (Lee et al., 2010a). The deoxyinosine nucleotide was removed from the nicked DNA by the 30 exonuclease activity of DNA polymerase I. Comparison of the amino acid sequences of EndoV from a set of diverse species reveals residues characteristic of this protein family (Fig. 2). These include the near fully conserved Asp43, Tyr80, Glu89, Asp110, His116 and Lys139 residues (numbers corresponding to the Tma enzyme). These residues are involved in catalysis, substrate or product binding, as described in detail in Section 3.1, and mutation of several of these residues in Tma EndoV has a major impact on the incision activity (Huang et al., 2002). Enzymes within the structurally and functionally diverse RNaseH-like superfamily depend on two divalent metal ions for catalysis, preferably Mg2þ, but Mn2þ can also support catalysis (Cherepanov et al., 2011; Davies et al., 1991; Maertens et al., 2010; Nowotny and Yang, 2006; Yang et al., 1990, 2006; Yang, 2011). The mechanism, in which one metal ion activates the attacking water molecule or sugar hydroxyl while the other ion coordinates and stabilizes the oxyanion leaving group, was first suggested for RNA-catalysed reactions involved in RNA splicing and RNase P cleavage of precursor tRNA (Steitz and Steitz, 1993). The two metalion catalysis has also been observed for the HH16 hammerhead ribozyme (Lott et al., 1998). Analyses of the metal dependent incision reveal that E. coli EndoV requires Mg2þ, but the divalent metal ion can at least partly be replaced with Mn2þ or Co2þ (Gates and Linn, 1977b; Yao et al., 1994; Yao and Kow, 1997). Similarly, Tma EndoV functions with both Mg2þ and Mn2þ (Feng et al., 2006; Lin et al., 2007), and it has been proposed that regulation of the catalysis can be controlled by binding of two metal ions, and also that Ca2þ may stimulate the Mn2þ mediated activity by binding to a second site (Feng et al., 2006). On the other hand, Ca2þ can not act as the sole metal ion in catalysis (Yao et al., 1994). Structural studies have only identified

one distinct metal binding site (Dalhus et al., 2009a), discussed in detail in Section 3.1, but this metal-containing structure is that of the product DNA complex after cleavage. Tma EndoV, using Mn2þ as cofactor, gives some unspecific cleavage of DNA for an oxanine (deaminated guanine) containing substrate, compared to the Mg2þ catalysed reaction (Hitchcock et al., 2004). Unspecific product formation was also observed with a hypoxanthine substrate. Mn2þ ions have also been observed to rescue the activity of several single-residue mutants of Tma EndoV with low activity when tested with Mg2þ on a hypoxanthine substrate (Feng et al., 2006). The underlying mechanism for this phenomenon is not clear, but it may have to do with the way in which Mn2þ is coordinated in the mutant enzymes. Although EndoV incises DNA at a variety of lesions, genetic analysis of an E. coli nfi mutant shows an increase in the frequency of nitrate-, nitrite- and nitrous acids-induced mutations in A:T base pairs, suggesting a role for EndoV in the in vivo repair of deaminated purine bases like hypoxanthine (Guo and Weiss, 1998; Schouten and Weiss, 1999; Weiss, 2001). Another study by Weiss suggests that E. coli EndoV initiates a nucleotide incision repair pathway responsible for removal of the majority of hypoxanthine in DNA (Weiss, 2008). Comparison of the repair of hypoxanthine by bacterial cell-free extracts with and without EndoV shows a 7-fold reduction in repair for the EndoV deficient extract (Lee et al., 2010a). On the other hand, a recent study showed no increased level of hypoxantine in genomic DNA in an E. coli nfi mutant (Pang et al., 2012), which questions the role of EndoV as a DNA repair protein involved in removal of hypoxanthine. In E. coli, the DNA glycosylase AlkA is shown to remove hypoxanthine (Saparbaev and Laval, 1994), a property also shared with the human alkyladenine DNA glycosylase AAG (Saparbaev and Laval, 1994; O'Brien and Ellenberger, 2003). 2.2. Mammalian EndoV e ribonuclease activity The EndoV homologues are highly conserved and present in all domains of life, though with exceptions for insects, protozoans and all fungi other than Schizosaccharomyces pombe (Fladeby et al., 2012). A multiple-sequence alignment of selected prokaryotic and eukaryotic EndoVs shows a highly conserved core domain, with variations essentially only at the C-terminal end (Fig. 2), where the mammalian versions, particularly mouse and rat EndoV, have ca 50e70 additional residues compared with other eukaryotic EndoVs, and more than 100 residues extra compared with prokaryotic EndoVs. The function of EndoV in mammalian cells is still not resolved. Several studies report no incision activity for human EndoV on various DNA substrates typical for prokaryotic EndoV (Fladeby et al., 2012; Vik et al., 2013; Morita et al., 2013), however one study reports on incision activity for deaminated adenine in DNA (Mi et al., 2012), and another study report only very weak incision activity for mouse EndoV (Moe et al., 2003). Human EndoV is able to form protein-DNA complexes with oligomers representing various DNA structures containing a discontinuous/distorted helix, such as 50 - and 30 -flaps, 3- and 4-way junctions, fork and pseudo-Y structures (Fladeby et al., 2012), a property shared with E. coli EndoV (Fladeby et al., 2012). Nonetheless, no incision activity was found for human EndoV on any of these substrates, in contrast to E. coli EndoV (Yao and Kow, 1996). Indeed, while no robust activity for mammalian EndoV on DNA containing hypoxanthine have been demonstrated (Fladeby et al., 2012; Mi et al., 2012; Moe et al., 2003; Morita et al., 2013; Vik et al., 2013), two recent studies reported the discovery of ribonuclease activity for human EndoV on inosine in RNA (Morita et al., 2013; Vik et al., 2013). Human EndoV, using Mg2þ or Mn2þ as the

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Fig. 2. Multiple-sequence alignment of EndoV sequences from a selection of prokaryotic and eukaryotic species. Key residues are indicated, using the numbering scheme from Tma EndoV. Sequences were retrieved from NCBI RefSeq, Uniprot (mouse) or Genbank (frog) (accession codes in parenthesis), aligned with ClustalW2, and the alignment was visualized using JalView (Waterhouse et al., 2009). Eubacteria: Tma ¼ Thermotoga maritima (NP_229661.1); Tth ¼ Thermus thermophilus (YP_004951.1); Eco ¼ Escherichia coli (NP_290630.1); Ent ¼ Enterobacter sp. (YP_001174952.1); Sty ¼ Salmonella typhimurium (NP_463037.1). Archea: Pis ¼ Pyrobaculum islandicum (WP_011762440.1); Afu ¼ Archaeoglobus fulgidus (NP_068968.1); Pfu ¼ Pyrococcus furiosus (NP_578716.1). Eukaryotes: Ehi ¼ Entamoeba histolytica (XP_647849.1); Spo ¼ Schizosaccharomyces pombe (NP_594332.1); Ath ¼ Arabidopsis thaliana (NP_567868.1); Xtr ¼ Xenopus tropicalis (AAH87745.1); Mmu ¼ Mus musculus (Q8C9A2.2); Rno ¼ Rattus norwegicus (EDM06795.1); Hsa ¼ Homo sapiens (NP_775898.2).

metal cofactor, is able to incise single-stranded and to a lesser extent double-stranded RNAs containing a single inosine nucleotide. Of particular interest is that cleavage was also observed for a sequence in the Gabra-3 transcript of the GABAA neurotransmitter, as well as transfer RNAs with inosine in the wobble position. These deaminated adenines are formed enzymatically by ADAR and ADAT enzymes, respectively, to change the coding information or to allow/enhance translation (Agris et al., 2007; Basilio et al., 1962; Orlandi et al., 2012; Su and Randau, 2011). The incision takes place at the second phosphodiester on the 30 side of inosine, similar to prokaryotic EndoV on DNA, and alanine mutations of the

conserved Asp52, Tyr91 and Glu100 in human EndoV abolish the incision activity (Morita et al., 2013; Vik et al., 2013). E. coli EndoV is also able to incise RNA substrates containing inosine (Vik et al., 2013). 3. Endonuclease V e structures and recognition of substrates Structures of enzymes in complex with their substrates can reveal the molecular basis governing substrate recognition and specificity, catalysis and regulation of activity. To date, three crystal structures of Tma EndoV in complex with different DNAs have been

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solved (Dalhus et al., 2009a; Rosnes et al., 2013). The structure of Tma EndoV without DNA substrate has been deposited in the PDB structural database (PDB id 3HD0), as has EndoV from Bacillus subtilis (PDB id 3GA2) and Streptomyces avermitilis (PDB id 3GOC). For eukaryotic EndoV, only the structure of unliganded human EndoV has been settled (Zhang et al., 2014). 3.1. Prokaryotic EndoV in complex with deaminated DNA The first 3D structures of an EndoV enzyme were those of the wild-type and the inactive D43A mutant of Tma EndoV in complex

with DNA containing a deaminated adenine (Dalhus et al., 2009a). The structures revealed several distinct features of the EndoV enzyme. Tma EndoV is an aba protein with a central 8-stranded bsheet of parallel and anti-parallel strands flanked on both side by ahelices (Fig. 3a). The first 5 b-strands, forming a 3-2-1-4-5 ribonuclease H-like structural module, flanked on one side by the first 3 N-terminal helices in EndoV, are common to enzymes in the large RNase H-like family (Majorek et al., 2014). This fold is thought to be one of the earliest protein folds (Ma et al., 2008), and is found in the core of enzymes such as E. coli RNaseH (Katayanagi et al., 1990; Yang et al., 1990), E. coli and yeast Holliday junction resolvases

Fig. 3. Structural aspects of prokaryotic EndoV. (a) Cartoon of the Tma EndoV backbone, showing the RNaseH-like fold with a central 3-2-1-4-5 b-sheet surrounded by a-helices. (b) Molecular surface of Tma EndoV with ligand DNA (blue stick model) showing the spatial organization of key structural elements. The strand-separating PYIP wedge (dark grey) protrudes from the protein surface to form a base binding pocket, into which the deaminated (Hx) base (red) is flipped. The double-headed arrows show the directions of the DNA helix. Positions of the active site and the scissile bond indicated with arrows. (c) Protein-DNA hydrogen bond contacts in the base lesion pocket. (d) Close-up view of the active site architecture in wild-type Tma EndoV showing the DNA incision, metal coordination and 3' and 5' end fixation by residues Asp43, Asp110, Lys139 and His214. The latter residue is Asp in many species, including most eukaryotes and E. coli. The positions of the bases for the two nucleotides are indicated with cyan spheres. (e) Stereo view of overlay of Tma EndoV in complex with loop DNA (light blue sticks) and hypoxanthine DNA (light brown sticks). The base binding pocket and wedge are shown as dark red and grey surfaces, respectively. The extra thymine in the loop DNA (T insert) replaces a base pair in the hypoxanthine substrate, shifting the next base pair several angstroms.

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RuvC and Ydc2, respectively (Ariyoshi et al., 1994; Ceschini et al., 2001), the catalytic domain of E. coli DNA transposase (Davies et al., 2000), the PIWI domain of P. furiosus Argonaute (Song et al., 2004), and the 50 endonuclease domain of the nucleotide excision repair protein UvrC from T. maritima (Karakas et al., 2007). The structural and evolutionary aspects of the RNaseH like

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superfamily have recently been summarized in an extensive bioinformatics analysis (Majorek et al., 2014). The enzyme binds to the minor groove of DNA, which is heavily distorted and bent close to 90 , and the deaminated nucleotide is flipped via the DNA minor groove into a nucleotide binding pocket (Fig. 3b and e). The hypoxanthine is recognized by a series of polar

Fig. 4. Structural aspects of eukaryotic EndoV. (a) Comparison of backbone conformation of human and Tma EndoV, showing the additional a-helix in human EndoV (green). The numbers refer to the b-strand order in the RNase H-like motif. (b) Close-up view of the wedge motif in human (blue) and Tma (gray) EndoV, showing the slight difference in conformation. Numbering corresponds to the Tma EndoV sequence. Corresponding residues in human EndoV are Pro90, Tyr91, Val92 and Ser93. (c) Protein surfaces of Tma (gray) and human (blue) EndoV, with wedges in darker shadings, showing the difference in surface contours of the homologous enzymes. The DNA (yellow ball-and-stick with Hx base in red) binding to human EndoV is modelled from the Tma EndoV structure by superposition of the protein backbone atoms.

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interactions involving backbone atoms in the enzyme (Fig. 3c). However, the exact hydrogen bond arrangement is not known, and the base may bind in a tautomeric or charged form (Dalhus et al., 2009a). The pocket is narrow and each side of the flipped base is interacting with a series of hydrophobic residues via van der Waals forces. The highly conserved motif PYIP, which includes the invariant Tyr80 and is located in the loop between b-strand 3 and a-helix 2, forms a surface-exposed wedge that separates the two DNA strands and forms part of the nucleotide binding pocket (Fig. 3b and e). The principle of DNA strand separation by use of 3e4 residues forming a loop wedge is also seen in other complexes, such as the human UV DNA-damaged binding complex (UV-DDB) (Scharer and Campbell, 2009; Scrima et al., 2008). In contrast, DNA glycosylases and mismatch repair binding proteins MutS/MutSa/MutSb use single residues as smaller ‘wegde’ or ‘finger’ motifs to intercalate and stabilize the DNA helix (Dalhus et al., 2009b). A conserved Asp-Glu-Asp motif coordinates the divalent Mg2þ ion in the active site next to the nucleotide binding pocket. Several residues form a physical barrier between the base lesion pocket and the active site, explaining the unusual incision at the second phosphodiester 30 to the deaminated base. This contrasts the endonuclease activity of bifunctional DNA glycosylases and AP endonucleases, which incise DNA at the damaged nucleotide (Dalhus et al., 2009b). In the structure of the wild-type Tma EndoV, the DNA substrate is incised, and the 30 and 50 ends of the cleaved product are firmly held in place by Mg2þ, Lys139 and His214 (Fig. 3d). There is only one metal ion in the crystal structure of wildtype Tma EndoV in complex with the incised product. The interactions between the free DNA ends and Mg2þ, Lys139 and His214 of EndoV make sure the enzyme binds tightly to the cleaved product, suggesting a coordinated hand-off of the intermediate to the next enzyme in the pathway, as observed for DNA glycosylases in the BER pathway (Mol et al., 2000; Wilson and Kunkel, 2000). It is interesting to note that His214 is replaced by the negatively charged Asp amino acid in several species, including E. coli and mammalian EndoV (Fig. 2). 3.2. Prokaryotic EndoV in complex with loop DNA Building upon data showing that prokaryotic EndoV also incises base mismatches and insertion/deletion (I/D) mismatch loops (Huang et al., 2001; Yao and Kow, 1994, 1996), a structure of Tma EndoV in complex with a DNA substrate containing a þ1 nucleotide loop has been investigated (Rosnes et al., 2013). The structure reveals that a normal nucleobase (in this case adenine) is flipped into the lesion recognition pocket and that the extra nucleotide in the loop rests on the surface of the strand-separation wedge (Fig. 3e). The incision thus takes place at the shorter strand, at the second phosphodiester bond 30 to the flipped base, in line with biochemical data. The novelty of this structure is the evidence that EndoV can flip normal DNA bases into the nucleotide binding pocket, abeit only in context of a weak point in the DNA, like a base mismatch, loop or other distortion. Biochemical data shows that mismatches with purines are better substrates than pyrimidines (Huang et al., 2001; Yao and Kow, 1994), which may possibly be attributed to the larger contact surface area for flipped purines when interacting with the hydrophobic walls of the base pocket. 3.3. Structural basis for RNA incision by human EndoV Recently, a crystal structure of human EndoV was presented (Zhang et al., 2014). The structure, which does not contain the RNA substrate, reveals an overall fold very similar to the prokaryotic EndoV. The largest difference is the presence of an additional a-

helix in the C-terminal part of human EndoV (Fig. 4a). There are also some differences in loop conformations between the prokaryotic and eukaryotic EndoV. For the key motif, the strand-separating wedge, the difference is rather small (Fig. 4b). An analysis of the enzyme surface reveals a more open nucleotide binding cleft in eukaryotic EndoV compared with Tma EndoV, and a straightforward superposition of the two molecules reveal that the DNA conformation in Tma EndoV is not compatible with the surface of human EndoV (Fig. 4c). For instance, the very favourable stacking between the DNA base 30 to hypoxanthine and the fully conserved Tyr residue in the wedge is lacking in this simple model. Without a structure of human EndoV in complex with RNA, it is not possible to conclude if the difference in wedge conformation is due to induced fit in the Tma EndoV structure. However, comparison with the unpublished structure of apo Tma EndoV (PDB id 3HD0) reveals that the loop conformation is very similar between the free and DNAbinding forms of Tma EndoV. There are no larger conformational rearrangements in Tma EndoV upon binding of DNA; the two protein chains superimpose with an rmsd of 0.5 Å for all Ca atoms. The differences in surface features between the Tma and human EndoV (Fig. 4c) make it hard to rationalize the structural basis for RNA incision of human EndoV compared to DNA incision for Tma EndoV. Biochemical experiments show that human EndoV can incise a single-stranded DNA, and also a double-stranded DNA to lesser extent, containing a single ribonucleotide 30 to the inosine (Morita et al., 2013; Vik et al., 2013). Hence, the 20 hydroxyl group in this particular position in the oligonucleotide seems to be critical and sufficient for incision activity. Analysis of the Tma EndoV-DNA and human apo EndoV structures suggest that a ribonucleotide in this position will have its additional hydroxyl group close to the conserved catalytic glutamate and/or water molecules surrounding the catalytic Mg2þ ion. However, this model is based on the DNA conformation in the Tma EndoV-DNA complex, which may be different in human EndoV both with respect to ribose puckering and the ribose-phosphate backbone conformation. In particular, the observed differences in binding surfaces between the Tma and human EndoV combined with the unknown conformation of the RNA and DNA/RNA hybrid substrates make it hard to pinpoint the structural basis for this specific requirement for catalysis. Also, the structural basis for the tRNA loop incision (Vik et al., 2013) can only be resolved by determining the atomic resolution structure of a protein-RNA complex. 4. Conclusions Prokaryotic EndoV has been known for long time to incise DNA 30 to a variety of base lesions, including deaminated adenine, guanine and cytosine, and EndoV may be involved in repair of these deaminated bases. The crystal structures of EndoV in complex with DNA provide a detailed insight into a mechanism of base recognition and strand incision unlike other DNA base processing enzymes such as DNA glycosylases and AP endonucleases. In contrast to DNA glycosylases that remove bases in DNA by direct excision, EndoV cleaves the DNA backbone at the second phosphodiester 30 to the deaminated base without removing the base itself. The enzyme uses a wedge motif to separate the DNA strands at the lesion, and flips the deaminated base into a tight pocket next to the active site. The enzyme binds tightly to the nicked product, suggesting a protective role and handoff of the strand-break product as one step in a coordinated pathway. However, the downstream pathway of EndoV incision still remains unresolved, as is also the dynamics of EndoV enzymes in searching for deaminated bases in DNA. Recent data demonstrating incision at inosine in RNA suggest a role for EndoV enzymes in processing and editing of RNAs. It appears like the prokaryotic EndoVs are able to incise both deaminated DNA and

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