Unique 5′-P recognition and basis for dG:dGTP misincorporation of ASFV DNA polymerase X

Fig 1. Sequences and secondary structures of DNA molecules captured in the complex structures. DNA1, DNA2, and DNA3 are self-complementary and form duplex via the sequences highlighted with underlines. DNA2 also form dG:dG Hoogsteen pairs at both ends. The red 2′,3′-dideoxy T (ddT) and 2′,3′-dideoxy A (ddA) residues are incorporated into the strands during crystallization.

Fig 2. Unique DNA binding mode. (A) Interactions between DNA and protein observed in the AsfvPolX:DNA1 structure. The protein is shown as a cartoon with the palm and finger domains colored in cyan and white, respectively. The template strand is shown as stick in atomic color (C, yellow; N, blue; O, red; P, orange). The primer strand is omitted for clarity. (B) Detailed interactions between AsfvPolX and the individual nucleotides. The N and O atoms are colored in blue and red, respectively, for both strands, whereas, the C and P atoms are colored in yellow and pink and green and orange for the template strand and primer strand, respectively. Water molecules are shown as red spheres. (C) Schematic representation summarizing the interactions between AsfvPolX and DNA1. The direct interactions (including H-bonds and electrostatic interactions) and the water-mediated interactions are indicated by the dashed lines in red and blue, respectively.

Fig 3. 5′-P of downstream oligo facilitates the productive complex assembly. (A) Sequence of 1nt-gap DNA4 and the overall structure of AsfvPolX:1nt-gap DNA4 complex. AsfvPolX is shown as a cartoon with the palm and finger domains colored in cyan and white, respectively. The template strand, primer, and downstream oligo are shown as stick with the C atoms colored in green, yellow, and yellow, respectively. The template residue (G8) is indicated with arrow. (B) Sequences of 2nt-gap(P) DNA5 and 1nt-gap(P) DNA5. (C) Overall structure of AsfvPolX:1nt-gap(P) DNA5:dGTP. AsfvPolX is shown as cartoon with palm and finger domains colored in cyan and white, respectively. DNA is shown as sticks with the C atoms colored in yellow, green, and green, for the template strand, primer, and downstream oligo, respectively. dGTP is also shown as sticks, Mn2+ ions are shown as red spheres.

Fig 4. Structural comparison between AsfvPolX and the homologous proteins. (A) Close-up view showing the DNA conformation and the kink between C9 and T10 of the template strand observed in AsfvPolX:1nt-DNA(P) DNA5:dGTP structure. Superposition of AsfvPolX:1nt-gap(P) DNA5:dGTP structure with (B) the NMR AsfvPolX:DNA:dGTP structure, (C) the HsPolβ structure, and the (D) TtPolX structure. The comparison is done based on both the palm and finger domains. For clarity, only the finger domain of AsfvPolX:1nt-gap(P) DNA5:dGTP is shown (as a cartoon in light blue). The 8-KD domains of HsPolβ and TtPolX are shown as a cartoon in wheat. In B-D, the template strand, primer, and downstream oligo of the AsfvPolX:1nt-gap(P) DNA5:dGTP structure, are colored in yellow, green, and green, respectively. DNA molecules in the NMR AsfvPolX structure (B) and the TtPolX structure (D) are colored in red. For the HsPolβ structure (C), the template strand is colored red and primer and downstream oligo are colored in blue. 5′-P is shown as sticks in all structures.

Fig 5. 5′-P recognition by AsfvPolX. (A) Surface presentation of the 5′-P binding pocket. (B) Detailed conformations of 5′-P and interacting residues. AsfvPolX is shown as a cartoon in white. Both 5′-P and the interacting residues are shown as sticks, with C atoms colored in green. The 2Fo-Fc map is contoured at 1.5 σ level. (C) Structural superposition of AsfvPolX:1nt-gap(P) DNA5:dGTP and AsfvPolX:DNA1 showing the conformational differences in the presence and absence of 5′-P. The color scheme of AsfvPolX:1nt-gap(P) DNA5:dGTP is identical to (B). For the AsfvPolX:DNA1 structure, the finger domain is shown as a cartoon in yellow; Arg125, Thr166, Arg168, and Leu174 are shown as sticks, with C atoms colored in magenta. The H-bonds are indicated by dashed lines in black and red in the two structures, respectively. (D) Sequence of DNA G31. (E) Isothermal titration calorimetry (ITC) analysis results showing the impacts of Arg125 and/or Arg168 mutations on DNA G31 binding (see S1 Data). (F) Quantification and comparison of in vitro dGTP misincorporation against DNA G31. The reactions are catalyzed by WT AsfvPolX, R125A, R168A, and R125/168A (see S1 Data). The data represent the mean of three…

Fig 6. The impacts of the H115-Arg127 platform on the dG:dGTP misincorporation. The dC:dGTP and dG:dGTP base pairs observed in (A) AsfvPolX:1nt-gap(P) DNA5:dGTP structure and (B) AsfvPolX:1nt-gap(P) DNA6:dGTP structure, respectively. The 2Fo-Fc maps are contoured at 1.5 σ level. (C) Quantification and comparison of in vitro dG:dGTP misincorporation assay catalyzed by WT AsfvPolX, H115D, H115E, H115F, R127A, and H115F/R127A mutants (see S1 Data). The data represent the mean of three independent experiments with SD values indicated by error bars. The dG:dGTP and dT:ddA base pairs observed at the insertion and postinsertion sites of (D) H115F/R127A:1nt-gap(P) DNA6:dGTP and (E) H115F:1nt-gap(P) DNA6:dGTP, respectively. (F) Structural comparison showing the conformational differences between AsfvPolX:1nt-DNA(P) DNA6:dGTP and H115F:1nt-gap(P) DNA6:dGTP. For clarity, the insertion site dG:dGTP base pairs and the AsfvPolX protein in AsfvPolX:1nt-gap(P) DNA6:dGTP structure are omitted. The C atoms of Phe115, Arg127, and the postinsertion site dT:ddA of H115F:1nt-gap(P) DNA6:dGTP are colored green in both (E) and (F), whereas, the C atoms are colored white for His115, Arg127, and for the…

Fig 7. Val120 and Leu123 affect dNTP binding and dG:dGTP misincorporation. Interactions between Val120 and the nucleobase of dG observed in (A) the AsfvPolX:1nt-gap(P) DNA6:dGTP and (B) the AsfvPolX:1nt-gap(P) DNA5:dGTP structures, respectively. AsfvPolX is shown as a cartoon in white. dG, dGTP, His115, and 117TGPV120 regions are shown as sticks in atomic colors (C, magenta; N, blue; O, red; P, orange). The 2Fo-Fc map (contoured at 1.5 σ level) is shown as cyan mesh. (C) Hydrophobic interaction between Leu123 and the nucleobase of dGTP observed in the AsfvPolX:1nt-gap(P) DNA6:dGTP structure. (D) ITC analysis results showing the impacts of Val120 and Leu123 on the dGTP binding (see S1 Data). (E) Quantification and comparison of in vitro dG:dGTP misincorporation assay catalyzed by WT AsfvPolX, V120A, and L123A mutants (see S1 Data). The data represent the mean of three independent experiments with SD values indicated by error bars.