- Research article
- Open Access
Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA Glycosylase
BMC Structural Biology volume 15, Article number: 10 (2015)
Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized.
This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. Comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms.
The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.
Repair of damages in DNA is an essential cellular process regulated by specialized molecular machinery. Base excision repair (BER) pathway  for repair of small lesions is initiated by monofunctional DNA glycosylases. These enzymes use a water molecule as a nucleophile to cleave the N-glycosidic bond between the target base and deoxyribose, releasing the damaged base and leaving an apurinic/apyrimidinic (AP) site . The BER pathway for the removal of uracil (Ura), which arises in DNA from deamination of cytosine (Cyt) or incorporation of dUTP during DNA synthesis, is initiated by uracil-DNA glycosylases (UDGs) . UDGs are divided in different families based on their substrate specificity . Members of family I-V share similar structure and motifs. Family I UDGs, which are also known as UNGs, specifically excise uracil from single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with the preference ssU > dsU:G > dsU:A . UNGs are ubiquitous enzymes that use highly conserved motifs for DNA binding, uracil recognition and excision.
The reaction mechanism for UNGs has been elucidated from a series of elegant structural studies in which wild-type and catalytically inactive mutant UNGs were captured in the DNA-bound form at various stages of action [6–12]. For detection of uracil in DNA, UNGs use a ‘pinch-push-pull’ mechanism, which involves a multi-step base flipping process. In the first step, serine and proline residues in three different loops (the ‘Pro-rich loop’, the ‘Gly-Ser loop’ and the ‘Leu-intercalation loop’) lead to a slight bending of the DNA through compression (‘pinch’) of the backbone. In the second step, a conserved leucine residue of the ‘Leu-intercalation loop’ penetrates into the minor groove and the DNA becomes fully bent and kinked leading to flipping of the uracil base (‘push’). The uracil nucleotide interacts with the ‘uracil recognition pocket’ where cleavage of the glycosidic bond takes place, and in the final step, the leucine residue is retracted (‘pull’). Two catalytic residues, an aspartic acid and a histidine, are found invariant in all UNGs (Table 1).
UNGs of poxviruses are, however, exceptional and most diverse members of this family. The motifs used by these enzymes are fully conserved in all orthopoxviruses but differ significantly from their counterparts in other organisms (Additional file 1: Table S1) [4, 13–15]. More importantly, UNGs of poxviruses assume a novel and essential role in viral replication and serve as an intrinsic component of the replication machinery. In the prototypic poxvirus Vaccinia, UNG (known as D4) binds to another viral protein A20 to form the heterodimeric processivity factor. A20 also binds to the DNA polymerase E9 and thereby tethers D4 to E9 [15–20] and assembles the heterotrimeric (D4:A20:E9) core of the processive polymerase complex. In the absence of D4, Vaccinia E9 synthesizes only short (<10 nucleotides) stretches of DNA [18, 21]. This essential role of D4 in supporting processive DNA synthesis is not dependent on its catalytic (glycosylase) activity .
Crystal structures of D4 have been reported in the free form [13,22; 2owr 1 , Footnote 1 2owq, 3 nt7, 4dof, 4dog] and as a non-productive2 Footnote 2 uracil complex [14; 4lzb]. Three dimensional structure of D4 retains the overall fold of UNG proteins- a central 4-stranded parallel β-sheet with α-helices on both sides (Fig. 1). There are two additional β-sheets in D4 (residues 2-6/12-16 and 107-109/215-217) not seen in any other UNG (Figs. 1a, b). Although the UNG-specific motifs are very different, the architecture of the uracil-binding pocket in D4 is remarkably similar to other UNGs and five of the six residues forming the pocket are identical. These residues include the catalytic Asp68 and His181, which are appropriately positioned for binding uracil and cleavage of the glycosidic bond . Considering the unique properties of poxvirus UNGs, it should be highly interesting to understand how these proteins adapt to utilizing the altered motifs for binding DNA, and recognizing and excising uracil.
All crystal structures of free-D4 and the structure of the uracil complex feature a characteristic dimeric packing. Although specific protein-protein interactions between D4 subunits vary to some extent the dimer interface is very similar in these structures [13, 14, 22]. This homodimer interface is formed by residues located in the C-terminal β-strand of the central β-sheet in each subunit, a flexible loop/short helix (residues 161–173) and a long helix (187–206) located near the C-terminus. The first site, which is located in a loop connecting the two C-terminal β-strands, represents one of the most flexible areas in the D4 structures (Fig. 1a). At high protein concentration D4 utilizes the same interface for dimerization in solution. However, when A20 is present, very specific interaction at this surface leads to the formation of a D4:A20 heterodimer (Fig. 1c) [19, 20]. Cumulative structural and biochemical evidences indicate that in the processive DNA polymerase complex, D4 remains catalytically active and possibly continuously scans and removes uracil from newly synthesized DNA [18–20]. Details of D4’s ability to bind and scan DNA, its enzymatic function and substrate specificity have been reported [22, 23]. However, structural characterization of the DNA-binding site(s) on D4 is missing.
Here, we report the first detailed view of D4-DNA interactions from a crystal structure of D4 in complex with a non-specific undamaged dsDNA. The structure of this complex for the first time reveals the DNA-binding interactions by an UNG from the poxvirus family. It also represents the first three-dimensional structure of a UNG bound to a truly undamaged dsDNA and provides a representative snapshot of the initial interaction between UNG and DNA. We also present a structural comparison of D4 and human UNG (hUNG) for which crystal structures are available in the DNA-free state, in complex with uracil-containing DNA and in complex with a DNA containing an abasic site [6–8, 10].
Results and discussion
Overall quality of the D4-DNA complex structure
The D4-DNA structure was refined to 2.89 Å resolution with Rcryst and Rfree values of 21.6 and 26.5 %, respectively. The overall quality of the structural model is good as indicated by MolProbity scores  and map correlation coefficients (Table 2). The final model consists of two D4 subunits (chains A and B), a DNA double helix with 12 nucleotides in chain C and 10 in chain D, 4 glycerol (GOL) molecules of which one is in each active site and 118 solvent atoms. Glycerol molecules have been previously observed in the active site of D4 (2owq, 2owr) and E. coli UNG (3eug). Quality of electron density for residues 184–188 of the ‘Leu-intercalation’ loop was poor. Therefore, these five residues were not modeled. As a result, the discussion of the ‘Leu-intercalation’ loop for this structure is limited to interactions of loop residues 180–183. Also, side chain atoms of residues 189 and 190 (except for CB) are not included in the final model due to lack of electron density. These residues are located in a flexible area in crystal structures of D4 [13, 14].
In chain C all 12 nucleotides were modeled but only 10 could be modeled in chain D due to insufficient electron density. Of the 10 base pairs forming the DNA double helix, 9 show regular Watson-Crick (WC) base pairing interactions and one non-WC interaction (Fig. 2; Additional file 1: Table S2). In general electron density for the DNA is good for the central regions (C: nucleotides 5–8; D: nucleotides 23–26) that interact with protein residues as indicated by map correlation coefficients (0.92-0.96; average 0.94) and temperature factors (30.5-46.3 Å2; average 37.6 Å2). The overall quality of the electron density for the DNA double helix is displayed in Additional file 1: Figure S1.
A comparison of map correlation coefficients of the DNA nucleotides and the interface residues in D4 is presented in Additional file 1: Table S3. An electron density map (2mFo-DFc map contoured at 1.5σ level) for both D4-DNA interfaces (chains A and D; chains B and C) is displayed in Additional file 1: Figure S2.
As expected the DNA represents a B-form right-handed double helix. Puckering of the sugar ring for several nucleotides differs from the predominant C2’-endo conformation seen in B-DNA (Additional file 1: Table S4). Similar deviations in sugar puckering conformation have been observed in various protein-DNA complexes.
Assembly of D4-DNA complex
DNA parameters derived from the analysis using the w3DNA web server (http://w3dna.rutgers.edu/)  are presented in Additional file 1: Tables S2 and S4. Analyses of DNA-DNA, protein-DNA and protein-protein interfaces using the PDBePISA web server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html)  are shown in Table 3 and Additional file 1: Table S5.
Conformations of the two D4 chains in the complex are also very similar with an rmsd value of 0.15 Å for superposition of all 213 residues in A and B. Each D4 chain binds only to one DNA strand. D4 chain A binds to DNA strand D and D4 chain B binds to DNA strand C (Figs. 2a, b). The electrostatic potential distribution shown in Fig. 2c illustrates the charge complementarity of the DNA binding surface of D4 and the negatively charged phosphate backbone of the DNA strands.
D4 chain B interacts with nucleotides 5–8 (ACGT) in the central part of chain C while D4 chain A interacts with nucleotides 23–26 (AAAC) of chain D. D4 residues at the protein-DNA interface are Ile67, Pro71, Gly128, Glu129, Thr130, Lys131, Gly159, Lys160, Thr161, Asp162, Tyr180, His181 and Ala183 (Table 3 and Additional file 1: Table S5). Polar protein-DNA interactions with the phosphate backbone involve hydrogen-bonds (2.6-3.2 Å) with Lys131, Lys160, Thr161, Tyr180 and His181 from each D4 chain (Table 3; Fig. 2). DNA binding residues in the two D4 chains superimpose very well (rmsd 0.13 Å).
Distortion in the DNA chains C and D such as bending and kinking are small compared to those observed in complexes of hUNG with specific uracil-containing DNA [6–8, 10]. The length of the DNA (~37-38 Å) is comparable to the value of 39–41 Å expected for an ideal extended B-form DNA; the widths of the major and minor groove are also similar to those for ideal B-form DNA.
Incidentally, the DNA double helix extends in the unit cell through non-covalent interactions between the ends of the DNA helices generated by the space group symmetry in a head-to-tail fashion (Additional file 1: Figure S3). Similar extended packing through DNA-DNA contacts of dsDNA with sticky or blunt ends (head-to-head and head-to-tail fashion) has been observed in other protein-DNA structures [27, 28]. On the other hand this arrangement of DNA chains may be a result of crystal packing.
Both protein-DNA interfaces in the complex have similar interface areas and interface residues (Additional file 1: Table S5). Interactions between the two D4 chains in the complex are minimal and confined to residues Glu32, Val33, Ser35, Trp36, Arg39, Ser132, Ile135, Try136 and Lys139. There is a salt bridge between residues Glu32 and Arg39 and a stacking interaction between Trp36 of two subunits.
Conformational changes induced by DNA binding
Upon formation of productive DNA complexes, UNGs undergo an ‘open to close’ conformational transition [6–10]. Such a conformational transition is not expected in the present complex since the DNA in the complex does not contain uracil. D4 residues involved in binding DNA are localized in three structural areas: the extended DNA-binding loop (residues 126–132), the Gly-Ser loop (residues 159–162) and the Leu-intercalation loop (residues 180–187). These three regions are labeled in Fig. 3. Involvement of these sites in DNA-binding and the resulting structural changes in the protein are discussed below. Largest deviations in the DNA complex are noticed in the extended helix/loop segment (residues 164–174,s1 site) joining the two C-terminal strands of the central β-sheet, and in the Leu-intercalation loop (s2 site). These two regions show varying degrees of disorder in D4 structures and portions of these areas are missing from several final models. Therefore, we examined the electron density maps for D4 structures especially focusing on these areas. In the following discussion we used reference free-D4 structures in which completeness of the model in relevant regions of the protein was good and supported by electron density. We also used the structure of the D4:uracil complex (4lzb) for comparison . This structure contains 12 molecules in the asymmetric unit and the s1 and s2 sites could be modeled in 11.
Extended DNA binding loop (residues 126–132)
In the DNA complex the side chain amino group of Lys131 of each D4 subunit forms a strong hydrogen bond (2.6 and 2.7 Å) with a phosphate oxygen atom of the partner DNA strand. However, there is no movement in this region of D4 upon DNA binding. Main chain and side chain atoms of Lys131 and neighboring residues superimpose very well with corresponding atoms in free-D4 structures 4dof, 4dog and 4lzb. In all structures the Lys131 side chain extends towards the spatially close Gly-Ser loop and engages in hydrogen bonding with the carboxyl oxygen atoms of Asp162. As a result, the extended DNA-binding loop tilts slightly towards the Gly-Ser loop (Fig. 4). Quality of electron density for Lys131, Asp162 and areas adjacent to these residues is usually very good in these crystal structures (Additional file 1: Figure S4). In a previously published study, the Lys131Val mutant was found to be defective in processivity function . Since this mutant did not show glycosylase activity it was assumed to have a major conformational defect . Both Lys131 and Asp162 are strictly conserved in UNGs of the orthopoxviruses  and thus may play an important role in specialized functions of these viral UNGs, such as a component of the processivity factor. Since neither of these residues is conserved in other UNGs they are less likely to play any direct role in catalysis. On the other hand interactions of Lys131 with Asp162 on the spatially adjacent helix/loop may be important for the stability of the local structure. Therefore the loss of glycosylase activity in the valine mutant may be due to structural impairment. At the same time, D4 surface near Asp162 is critical for its interactions with A20  and therefore, structural changes due to the loss of interactions between Lys131 and Asp162 may affect the processivity function.
In a study combining H/D exchange mass spectroscopy and computational docking Roberts et al.  identified two extended non-sequence-specific DNA-binding surfaces in hUNG (residues 210–220 and 251–264) that did not show contacts with DNA in the crystal structures. In D4 the corresponding regions are residues 126–136 and 165–178. The latter segment in D4 is engaged in homodimer formation in the absence of A20 and in heterodimer formation in the presence of A20 . Thus this (residue 165–178) may not represent a potential DNA-binding site in D4.
Gly-Ser loop (residues 159–162)
The s2 site encompasses the Gly-Ser loop. This region shows varying degrees of flexibility in different D4 structures but could be modeled in both subunits in the DNA complex. Quality of electron density in this area of D4 was sufficient for unambiguously placing main chain and side chain atoms of all residues. For comparison of this site we selected the free-D4 structure 4dof (chains A, B) and the D4:uracil complex (4lzb, chain D, F). Comparison of the s2 site in the DNA-complex with free-D4 structures reveals a slight reorganization in this area of the molecule (Fig. 5). Two amino acid residues, Lys160 and Thr161, are involved in hydrophilic interactions with the DNA. Main chain nitrogen atom of Lys160 interacts with two oxygen atoms on one phosphate group (Table 3). Thr161 hydroxyl oxygen atom is hydrogen bonded to one of the oxygen atoms of the same phosphate also through the peptide nitrogen atom. Notably, Lys160 and Thr161 are unique residues in poxvirus UNGs and are replaced by glycine and serine respectively in both E. coli and human enzymes. We showed that D4 mutant Lys160Val was deficient in processivity function but retained catalytic activity and DNA binding ability . In all D4 structures, side chain of Lys160 extends toward the neighboring β-strand and its NZ atom forms a hydrogen bond with the peptide oxygen atom of Val178 on this strand. Loss of this contact in the Lys160Val mutant may impact the structure of this area of D4. Notably, a hydrophobic pocket at this site has been shown to be important for D4’s interaction with A20 .
Leu-intercalation loop (residues 180–187)
The s3 area, which includes the ‘Leu-intercalation loop’, is important for the catalytic mechanism. Electron density for this segment of D4 structure in 4dog and 3 nt7 was good . In the DNA-complex quality of electron density for this loop was inadequate for modeling the entire loop. Therefore, residues 184–188 were omitted from the final model. Two amino acid residues immediately preceding the disordered region of D4, namely Tyr180 and His181, directly interact with DNA. Side chain hydroxyl group of Tyr180 and the main chain nitrogen atom of His181 form contact with oxygen atoms of two phosphate groups from the DNA (Fig. 6). Tyr180 is conserved in UNGs of poxviruses but not in other UNGs. His181 is a critical catalytic residue and is conserved in UNGs across the species. In 4zlb we noticed some deviations in the position of His181 in different subunits. Generally, in a productive uracil complex, the distance between the NE2 atom of histidine and the O2 atom of uracil is significantly shorter than in non-productive complexes [6–8]. In 4lzb, the distance between O2 atom of uracil and His181 NE2 atom varied from 2.8-4.8 Å in different subunits . In the DNA complex His181 side chain moved farther away from the active site area. The missing segment includes Arg185, which is equivalent to the conserved leucine residue (272 in hUNG) of the Leu-intercalation loop. In the mutational analysis mentioned above D4 Arg187Val mutant was also shown to be unable to support processive DNA synthesis but retained binding to A20 and DNA . Unfortunately, due to disorder in this loop detailed comparison of this functionally significant region is not possible from the present structure.
Movement of Arg167 and implications for A20 binding
Most significant changes in the DNA complex were noticed in residues Arg167 and Pro173 although neither of these residues is involved in direct interaction with DNA. In 4dof and 4lzb, orientation of the Arg167 side chain is stabilized by a strong hydrogen bond between its NH2 atom and the main oxygen atom of Thr176. In both D4 subunits in the DNA complex, Arg167 side chain is directed away from Thr176 (Fig. 7a). The Arg167 side chain is oriented such that nitrogen atoms of the terminal amino groups form hydrogen bonds with the peptide oxygen atom of Val174. Position of Arg167 side chain is further stabilized by polar interactions of its amino groups with the hydroxyl oxygen atom of Thr176 side chain on one side and the main chain oxygen atom of Ser172 on the other (Fig. 7b).
In the crystal structure of the D4:A20 complex, the aromatic side chain of Trp43 of A20 occupies a hydrophobic pocket referred to as groove by Contesto-Richefeu et al.  and inserts into the space between Pro173 and Arg167 side chain. Movement of Arg167 side chain and Pro173 in the A20 complex (as compared to free D4 structures) favors this interaction. On the other hand in the DNA-complex, orientation of Arg167 side chain would interfere with binding of A20 at this site. Interestingly, movement of Pro173 in the DNA-complex also seems to create an unfavorable steric environment for binding of A20. While in the D4:A20 complex, Pro173 of D4 moves in a direction that allowed packing of Lys44 side chain of A20, in the DNA complex the carbonyl oxygen Pro173 would be in close contact (~1.5 Å) with the CB atom of Lys44 of A20 (Fig. 7c). Thus the structure of the present DNA complex suggests that DNA binding induces conformational changes in D4 that may interfere with its binding to A20. While D4 can independently bind both DNA and A20, perhaps in the formation of the heterotrimeric complex, D4-A20-interaction precedes the DNA-binding event.
Comparison of hUNG and D4
Crystal structures of hUNG have been reported in the free-enzyme form [1akz; 6] and in complex with damaged DNA containing uracil [1ssp; 6]. Structure of mutant hUNG in which the leucine residue (272) of the Leu-intercalation loop was altered to alanine was described in complex with DNA containing an abasic site [2ssp; 6]. Superimposition and comparison of the structure of hUNG with D4 in their DNA complexes demonstrate the divergences and general overlap in the areas important for DNA binding and UNG catalytic mechanism. The three DNA binding sites described above are shown in Fig. 8. Structural comparison of hUNG in the free-state and in complex with different DNA constructs demonstrated that major conformational changes in UNGs are promoted by the steps involved in generating and stabilizing the flipped out nucleotide upon recognition of uracil . Upon binding an undamaged DNA, these conformational changes are not expected in D4. Superimposition of the structure of the D4:DNA complex and the DNA complex of hUNG (1ssp) reveals that the Gly-Ser loop and the Leu-intercalation loop provide the main interactions with DNA. Part of the Leu-intercalation loop is disordered in the D4:DNA complex. In hUNG, the catalytic histidine (His268 in 1ssp) clearly moves slightly closer towards the uracil binding pocket as compared to the D4:DNA complex. It should be noted that in DNA-free state this histidine residue superimposes well in D4 and hUNG (see Fig. 8). The interactions between the DNA and D4 at the extended DNA-binding site may be facilitated because of the larger size of the bound DNA as compared to hUNG:DNA complexes.
The D4-DNA complex described here provides the structural framework for recognizing the DNA binding residues and motifs in poxvirus UNGs. The process of sequence-specific recognition and binding of DNA includes non-specific interaction as an early or intermediate step [30–33]. The structure with undamaged dsDNA may represent a snapshot of an early DNA-protein interaction in preparation for the recognition complex for UNG . It remains to be seen if the Leu-intercalation loop transitions from its disordered state to an ordered state upon recognition of a damage in the DNA. Some of the unique structural features of D4 have evolved for undertaking its novel role in viral replication. Structure presented here provides potential understanding of DNA-binding by D4 in the poxvirus replication machinery.
Preparation and crystallization of D4-DNA complex
Custom DNA oligos purified by standard desalting method were ordered from Integrated DNA Technologies (IDT). The 12mer DNA oligo was designed to be self-complementary: 5’-GCA AAC GTT TGC-3’. The DNA oligo was dissolved in annealing buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA) at a concentration of 5 mM. Annealing using a Perkin Elmer GeneAmp PCR System 2400 resulted in a blunt-end 12mer double-stranded DNA construct with 12 base pairs (final concentration 2.5 mM):
5’-GCA AAC GTT TGC-3’ (forward strand)
3’-CGT TTG CAA ACG-5’ (reverse complementary strand)
The following annealing protocol was used:
Heating to 368 K in 5 min;
Cooling to 338 K in 5 min;
Cooling to 328 K in 5 min;
Cooling to 318 K in 5 min;
Cooling to 298 K in 5 min;
Cooling to 277 K in 40 min.
DNA oligos and annealed dsDNA were kept at 253 K until use.
Protein solution at 3 mg/mL (~120 μM in 25 mM HEPES buffer, pH 7.3, 60 mM KCl, 1 mM TCEP) was incubated with previously annealed self-complementary non-specific dsDNA in a 1:1.2 molar ratio at 277 K for 1 h. High-throughput crystallization screening using the PEGs and Protein Complex Suites from Qiagen was conducted on a Crystal Gryphon (Art Robbins Instruments). Thin plate-like crystals were obtained in hanging drop vapor diffusion at 293 K. The drops contained 1 μL protein plus 0.5 μL reservoir solution containing 16 % PEG6K, 0.08 M TRIS buffer, pH 8.5, 20 % glycerol. For cryo-freezing, crystals were directly plunged into liquid nitrogen.
Data collection and refinement
Intensity data were collected on a Pilatus 6 M detector at the Advanced Photon Source on NE-CAT beamline 24-ID-C. Data were processed with XDS [36, 37] and SCALA  of the CCP4 program suite  as part of the RAPD data-collection strategy at NE-CAT (https://chem.cornell.edu/rapd/). A total of 120 images (1° width) were collected. While the first 75 frames showed overall good statistics frames 76–120 showed increased mosaicity and Rmerge values. The processed data from frames 1–75 were indexed in the orthorhombic crystal system. Probabilities for Laue group P222 and space group P212121 based on systematic absences were 0.856 and 0.967, respectively. Diffraction data extending to 2.89 Å resolution were used for molecular replacement and refinement. CC1/2 and CC* values at 2.89 Å resolution were 0.86 and 0.96, respectively [40, 41]. Data-collection statistics are listed in Table 1.
The unit-cell parameters (a = 39.4 Å, b = 92.3 Å, c = 142.9 Å; space group P212121) suggested two protein subunits in the asymmetric unit (52 % solvent; Matthews coefficient VM = 2.6). The crystal structure was solved by molecular replacement with Phaser (version 2.5.6)  using the coordinates of one subunit from the native D4 structure (4dof) as search model. The observed pseudo-translation vector was used to position the second subunit in the asymmetric unit. Of the alternative space groups in Laue group P222 only space group P212121 provided solutions for the translation function. The R value of the refined solution was 45.2 % (RFZ = 5.8, TFZ = 9.3, LLG = 1803). After 1 round of refinement (10 cycles) of the protein coordinates (Rcryst and Rfree dropped to 29.2 % and 32.9 %, respectively) in REFMAC (version 5.8.0073)  electron density for the DNA double helix became visible in sigmaA weighted 2mFo-DFc and mFo-DFc difference electron density maps (m is the figure of merit and D is the sigmaA weighting factor). Using modeling tools in Coot (version 0.8-pre)  B-form DNA nucleotides were fitted into the electron density maps. The forward strand (chain C) of the dsDNA is numbered from 1–12 (5’ to 3’) while the reverse complementary strand (chain D) is numbered 21–32 (5’ to 3’). Base pairing occurs between the base of nucleotide 1 and the base of nucleotide 32 and similarly between the nucleotide 12 and 21 (and so forth). For structure refinement we used automatically generated local non-crystallographic symmetry (NCS) restraints in REFMAC. Structure validation was accomplished using PHENIX (version 1.9-1692) [45, 46], MolProbity , QualityCheck (http://smb.slac.stanford.edu/jcsg/QC/) , and the new wwPDB X-ray validation server (http://wwpdb-validation.wwpdb.org/validservice/). PHENIX and REFMAC were used for final map calculations. Refinement statistics are listed in Table 1. The structure has been deposited in PDB under 4qcb. The difference between R and Rfree, although within reasonable limit for structures in similar resolution, is somewhat higher and may result from incompleteness of the models (for DNA and protein).
PDBID’s are shown in italics
Uracil in this complex is not a product of glycosylase activity
Vaccinia uracil-DNA glycosylase
Base excision repair
Family I uracil-DNA glycosylase
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This work was supported by the National Institutes of Health grant 5U01-A1-082211.
The work is based upon research conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, which are supported by a grant from the National Institute of General Medical Sciences (P41 GM103403) from the National Institutes of Health. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.
The authors declare that they have no competing interests.
NS was involved in crystallization, structure determination, structure refinement and manuscript preparation. SB performed data collection and data processing. RR and DC were involved in manuscript preparation. DC designed and participated in the experiments, and provided technical and scientific guidance. All authors read and approved the final manuscript.
Sequence alignment of six specific regions in UNG enzymes that include the motifs for DNA binding and catalysis. Table S2. Detailed hydrogen bonding information for DNA base pairing (w3DNA analysis) in 4QCB. Table S3. Map correlation coefficients and average B values for protein interface residues and DNA nucleotides. Table S4. Conformational sugar parameters in 4QCB (w3DNA analysis). Table S5. Analysis of protein-protein, protein-DNA and DNA-DNA interfaces in 4qcb (PISA analysis). Figure S1. SigmaA weighted 2mFo-DFc and mFo-DFc omit maps for DNA region. DNA double helix with chains C and D. A. 2mFo-DFc map (contoured at 1.5σ level). B. mFo-DFc omit map (contoured at 2.5σ level). C. 2mFo-DFc simulated annealing omit map (contoured at 1.0σ level). Figure S2. SigmaA weighted 2mFo-DFc map for D4-DNA interfaces. A. Stick model representation of D4-DNA interface 1 (chains A and D). B. 2mFo-DFc map of D4-DNA interface 1 (chains A and D). C. Stick model representation of D4-DNA interface 2 (chains B and C). D. 2mFo-DFc map of D4-DNA interface 2 (chains B and C). Figure S3. Packing of D4-DNA complex. Figure S4. Electron density map.
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Schormann, N., Banerjee, S., Ricciardi, R. et al. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA Glycosylase. BMC Struct Biol 15, 10 (2015). https://doi.org/10.1186/s12900-015-0037-1
- Protein-DNA structure
- Non-specific DNA
- Early DNA recognition complex
- Uracil-DNA glycosylase