Table 1.
Data collection and refinement statistics of mvUNG.
Fig 1.
(a) Overall domain organization of mvUNG. Green, blue, and red bars represent the relative lengths of the catalytic domain, N-terminal domain, and motif-I, respectively. The schematic shows that mvUNG contains a long N-terminal domain and motif-I, which is inserted in the catalytic domain. (b) Enzymatic activity of mvUNG. In the silver-stained gel, the top and bottom bands indicate uncleaved ssDNA containing a single uracil and cleaved fragments, respectively. This shows that mvUNG has the activity of uracil-DNA glycosylase activity. (c) Size measurement by SEC-MALLS. The molecular mass of purified mvUNG in solution was calculated as 43.4 kDa, which is close to that of the monomer (molecular weight of a monomer: 42.2 kDa).
Fig 2.
(a) Structure-based sequence alignment of UNGs. UNG sequences from Acanthamoeba polyphaga mimivirus (mvUNG), human (hUNG), herpes simplex virus (hsvUNG), vaccinia virus (vacvUNG) and Escherichia coli (EcUNG) are aligned based on a structural comparison. Identical and homologous residues are boxed in red and yellow, respectively. Residues of the active site are marked with blue triangles. The five conserved motifs in UNGs are boxed and labeled in blue. The secondary structure of mvUNG is displayed using a cylinders for helices and arrows for β-strands. Helices and β-strands are labeled with H and S, respectively. Helix labels with and without an asterisk (*) indicate an α-helix and 310 helix, respectively. (b, c) Ribbon models of mvUNG shown at two different orientations. α-helix, β-strand, and loops are colored differently, and the secondary structures are labeled using the same scheme as in (a). (d) Structural comparison of UNGs. Structures of mvUNG, hUNG (PDB ID: 1AKZ), and hsvUNG (PDB ID: 1UDG) were superimposed, and Cα trace models were drawn in the same orientation as (b). Red, yellow, and blue represent mvUNG, hUNG, and hsvUNG, respectively. Additional segments in mvUNG (motif-I and residues 95–130 of the N-domain) are colored green and labeled.
Fig 3.
(a) Motifs of mvUNG conserved in UNG proteins. The ribbon and stick model covered by a transparent yellow surface is shown. Residues of the water-activating, Pro-rich, uracil-recognition, Gly-Ser, and minor-groove intercalation motifs are labeled and colored green, blue, orange, purple, and magenta, respectively. Motif-I (black dotted oval) is on the left side of the Gly-Ser loop (purple) and the minor-groove intercalation loop (magenta) in this figure. (b) The DNA-binding model of mvUNG. The model was generated by superimposing mvUNG on the structure of hUNG/DNA containing an abasic site (PDB ID: 2SSP). mvUNG was drawn as a surface model with charge distribution. DNA in the structure of hUNG/DNA complex is drawn as a stick model. The figure shows that DNA is intercalated by Y322 and binds to a positively charged groove formed by five motifs conserved in UNG family proteins. mvUNG structures in (a, b) are in the same orientation as in Fig 2b. (c) Comparison of active site residues. Structures of mvUNG and hUNG (PDB ID: 1AKZ) are superimposed. Red and blue Cα trace models represent mvUNG and hUNG, respectively. Residues of the active site are drawn as sticks and labeled. Leu residue in minor-groove intercalation loop of hUNG is replaced by Tyr (Y322) in mvUNG.
Fig 4.
Activity comparison of mvUNG mutants.
(a) Silver-stained gel showing the cleavage of ssDNA by mvUNG mutants. Full-length mvUNG, mvUNGΔ327–343, mvUNG95-370, and mvUNG122-370 were incubated with ssDNA to compare their UNG activities. (b) Relative activities of mvUNG mutants drawn as a histogram. The activity was calculated based on the amount of remaining uncleaved substrate. Seven independent measurements were averaged (S6 Fig). P values were calculated using a two-sample t-test. Error bars indicate mean ± standard error (*P< 0.05, **P < 0.01, ***P < 0.001, n = 7). In the reaction of 0.2 μM mvUNG and 10 μM ssDNA, the activities of mvUNGΔ327–343, mvUNG95-370, and mvUNG122-370 were reduced to 59, 55, and 15% of the activity of full-length mvUNG, respectively. In the reaction of 1 μM mvUNG and 10 μM ssDNA, the activities of mvUNGΔ327–343, mvUNG95-370, and mvUNG122-370 were reduced to 73, 58, and 47% of the activity of the full-length mvUNG, respectively.
Fig 5.
Circular dichroism (CD) spectra of mvUNGs.
(a) Far-UV CD spectra of full-length mvUNG (Full) and mvUNG95-370 (95–370). The two CD spectra show similar patterns and ellipticity scales, indicating that residues 1–94 of mvUNG are close to a random coil conformation. (b) Thermal melting curves. Melting curves were collected at a wavelength of 220 nm by increasing temperature to 90°C. Calculated Tm values of full-length mvUNG, mvUNG95-370, mvUNG122-370, and mvUNGΔ327–343 were 55.9, 51.5, 48.7, and 46.5°C, respectively, indicating that deletion of the additional segments decreased the thermal stability of mvUNG.