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

<div><p>African swine fever virus (ASFV) can cause highly lethal disease in pigs and is becoming a global threat. ASFV DNA Polymerase X (<i>Asfv</i>PolX) is the most distinctive DNA polymerase identified to date; it lacks two DNA-binding domains (the thumb domain and 8-KD domain) conserved in the homologous proteins. <i>Asfv</i>PolX catalyzes the gap-filling reaction during the DNA repair process of the ASFV virus genome; it is highly error prone and plays an important role during the strategic mutagenesis of the viral genome. The structural basis underlying the natural substrate binding and the most frequent dG:dGTP misincorporation of <i>Asfv</i>PolX remain poorly understood. Here, we report eight <i>Asfv</i>PolX complex structures; our structures demonstrate that <i>Asfv</i>PolX has one unique 5′-phosphate (5′-P) binding pocket, which can favor the productive catalytic complex assembly and enhance the dGTP misincorporation efficiency. In combination with mutagenesis and in vitro catalytic assays, our study also reveals the functional roles of the platform His115-Arg127 and the hydrophobic residues Val120 and Leu123 in dG:dGTP misincorporation and can provide information for rational drug design to help combat ASFV in the future.</p></div

The impacts of the H115-Arg127 platform on the dG:dGTP misincorporation.

<p>The dC:dGTP and dG:dGTP base pairs observed in (<b>A</b>) <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP structure and (<b>B</b>) <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP structure, respectively. The 2F_o-F_c maps are contoured at 1.5 σ level. (<b>C</b>) Quantification and comparison of in vitro dG:dGTP misincorporation assay catalyzed by WT <i>Asfv</i>PolX, H115D, H115E, H115F, R127A, and H115F/R127A mutants (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). 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 (<b>D</b>) H115F/R127A:1nt-gap(P) DNA6:dGTP and (<b>E</b>) H115F:1nt-gap(P) DNA6:dGTP, respectively. (<b>F</b>) Structural comparison showing the conformational differences between <i>Asfv</i>PolX:1nt-DNA(P) DNA6:dGTP and H115F:1nt-gap(P) DNA6:dGTP. For clarity, the insertion site dG:dGTP base pairs and the <i>Asfv</i>PolX protein in <i>Asfv</i>PolX: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 (<b>E</b>) and (<b>F</b>), whereas, the C atoms are colored white for His115, Arg127, and for the postinsertion site dT:ddA of <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP in (<b>F</b>).</p

K_d values for the DNA binding to <i>Asfv</i>PolX and mutants.

<p>K_d values for the DNA binding to <i>Asfv</i>PolX and mutants.</p

5′-P of downstream oligo facilitates the productive complex assembly.

<p>(<b>A</b>) Sequence of 1nt-gap DNA4 and the overall structure of <i>Asfv</i>PolX:1nt-gap DNA4 complex. <i>Asfv</i>PolX 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>B</b>) Sequences of 2nt-gap(P) DNA5 and 1nt-gap(P) DNA5. (<b>C</b>) Overall structure of <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP. <i>Asfv</i>PolX 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.</p

Val120 and Leu123 affect dNTP binding and dG:dGTP misincorporation.

<p>Interactions between Val120 and the nucleobase of dG observed in (<b>A</b>) the <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP and (<b>B</b>) the <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP structures, respectively. <i>Asfv</i>PolX 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 2F_o-F_c map (contoured at 1.5 σ level) is shown as cyan mesh. (<b>C</b>) Hydrophobic interaction between Leu123 and the nucleobase of dGTP observed in the <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP structure. (<b>D</b>) ITC analysis results showing the impacts of Val120 and Leu123 on the dGTP binding (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). (<b>E</b>) Quantification and comparison of in vitro dG:dGTP misincorporation assay catalyzed by WT <i>Asfv</i>PolX, V120A, and L123A mutants (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). The data represent the mean of three independent experiments with SD values indicated by error bars.</p

5′-P recognition by <i>Asfv</i>PolX.

<p>(<b>A</b>) Surface presentation of the 5′-P binding pocket. (<b>B</b>) Detailed conformations of 5′-P and interacting residues. <i>Asfv</i>PolX 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 2F_o-F_c map is contoured at 1.5 σ level. (<b>C</b>) Structural superposition of <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP and <i>Asfv</i>PolX:DNA1 showing the conformational differences in the presence and absence of 5′-P. The color scheme of <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP is identical to (<b>B</b>). For the <i>Asfv</i>PolX: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. (<b>D</b>) Sequence of DNA G31. (<b>E</b>) Isothermal titration calorimetry (ITC) analysis results showing the impacts of Arg125 and/or Arg168 mutations on DNA G31 binding (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). (<b>F</b>) Quantification and comparison of in vitro dGTP misincorporation against DNA G31. The reactions are catalyzed by WT <i>Asfv</i>PolX, R125A, R168A, and R125/168A (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). The data represent the mean of three independent experiments, with standard deviation (SD) values indicated by error bars.</p

Structural comparison between <i>Asfv</i>PolX and the homologous proteins.

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

Unique DNA binding mode.

<p>(<b>A</b>) Interactions between DNA and protein observed in the <i>Asfv</i>PolX: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>B</b>) Detailed interactions between <i>Asfv</i>PolX 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. (<b>C</b>) Schematic representation summarizing the interactions between <i>Asfv</i>PolX 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.</p