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Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase.

Gaur V, Vyas R, Fowler JD, Efthimiopoulos G, Feng JY, Suo Z - Nucleic Acids Res. (2014)

Bottom Line: Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years.These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180° with an unusual O4'-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase.Such rare binding modes significantly decrease the incorporation rates and efficiencies of these L-nucleotides catalyzed by the polymerase.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.

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Related in: MedlinePlus

Difference in the stacking interactions of Tyr12 of Dpo4 with various incoming nucleotides. (A) Superimpositions of incoming D-dCTP, (–)3TC-PPNP, and Type-A and Type-B conformations of (–)FTC-PPNP in stereoview. Sulfur atoms at the 3′ positions in sugar rings are shown as spheres and (B) superposition incoming D-dCDP, L-dCDP (chains A and D), (–)3TCDP and (–)FTCDP in stereoview.
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Figure 3: Difference in the stacking interactions of Tyr12 of Dpo4 with various incoming nucleotides. (A) Superimpositions of incoming D-dCTP, (–)3TC-PPNP, and Type-A and Type-B conformations of (–)FTC-PPNP in stereoview. Sulfur atoms at the 3′ positions in sugar rings are shown as spheres and (B) superposition incoming D-dCDP, L-dCDP (chains A and D), (–)3TCDP and (–)FTCDP in stereoview.

Mentions: Interestingly, all three incoming L-nucleotides in the ternary structures were present in anti-conformation and formed Watson–Crick base pairs with the templating nucleotide dG (Figure 2). The triphosphate of (–)3TC-PPNP displayed a novel N-shaped conformation (Figure 2B), unlike the typically observed chair-like conformations (46) of matched D-dNTPs complexed with Dpo4 and undamaged DNA (Supplementary Table S2), e.g. Dpo4-D-dCTP (Figure 2A). In contrast, the triphosphate of (–)FTC-PPNP displayed two alternate conformations: the N-shaped conformation (Type-A, Figure 2C) and the goat tail-like conformation (46) (Type-B, Figure 2D). After several rounds of refinement with different occupancy ratios, the two conformations of (–)FTC-PPNP were best modeled with equal occupancies (Supplementary Figure S6). Although similar to (–)3TC-PPNP, the triphosphate moiety in Type-A conformation of (–)FTC-PPNP was bent slightly toward the primer 3′-terminus, allowing a water molecule to slide between Tyr12 and (–)FTC-PPNP (Figure 2C). The conformations adopted by the α- and β-phosphates in the N-shaped conformations of (–)3TC-PPNP and (–)FTC-PPNP are nearly identical to the equivalent α- and β-diphosphate moieties in Dpo4-(–)FTC-DP, Dpo4-(–)3TC-DP and Dpo4-L-dCDP (Figure 3 and Supplementary Figure S7) as well as the ternary structures of Dpo4·DNA·ddNDP (Supplementary Table S3), which display an Λ-shaped diphosphate conformation (Figure 4D). This supports our above conclusion that the (β,γ-imido)-substitution in (–)3TC-PPNP and (–)FTC-PPNP does not significantly affect the binding conformation of an incoming nucleotide. Moreover, it also indicates that either the absence of the 3′-OH of an incoming nucleotide or the mismatching of the nucleotide with a templating base significantly altered the binding conformations of the nucleotide and DNA at the active site of Dpo4. In contrast, the absence of the γ-phosphate moiety did not affect the conformation of an incoming nucleotide and the overall structure of a ternary complex, which was likely due to the positioning of the γ-phosphate moiety on the edge of the Dpo4 surface (Supplementary Figure S8A). Consistently, the nearly superimposable structures of Dpo4-D-dCDP and Dpo4-D-dCTP show that D-dCDP in Dpo4-D-dCDP is bound with an almost identical location and conformation as the counterpart of D-dCTP in Dpo4-D-dCTP (Supplementary Figure S7A).


Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase.

Gaur V, Vyas R, Fowler JD, Efthimiopoulos G, Feng JY, Suo Z - Nucleic Acids Res. (2014)

Difference in the stacking interactions of Tyr12 of Dpo4 with various incoming nucleotides. (A) Superimpositions of incoming D-dCTP, (–)3TC-PPNP, and Type-A and Type-B conformations of (–)FTC-PPNP in stereoview. Sulfur atoms at the 3′ positions in sugar rings are shown as spheres and (B) superposition incoming D-dCDP, L-dCDP (chains A and D), (–)3TCDP and (–)FTCDP in stereoview.
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Figure 3: Difference in the stacking interactions of Tyr12 of Dpo4 with various incoming nucleotides. (A) Superimpositions of incoming D-dCTP, (–)3TC-PPNP, and Type-A and Type-B conformations of (–)FTC-PPNP in stereoview. Sulfur atoms at the 3′ positions in sugar rings are shown as spheres and (B) superposition incoming D-dCDP, L-dCDP (chains A and D), (–)3TCDP and (–)FTCDP in stereoview.
Mentions: Interestingly, all three incoming L-nucleotides in the ternary structures were present in anti-conformation and formed Watson–Crick base pairs with the templating nucleotide dG (Figure 2). The triphosphate of (–)3TC-PPNP displayed a novel N-shaped conformation (Figure 2B), unlike the typically observed chair-like conformations (46) of matched D-dNTPs complexed with Dpo4 and undamaged DNA (Supplementary Table S2), e.g. Dpo4-D-dCTP (Figure 2A). In contrast, the triphosphate of (–)FTC-PPNP displayed two alternate conformations: the N-shaped conformation (Type-A, Figure 2C) and the goat tail-like conformation (46) (Type-B, Figure 2D). After several rounds of refinement with different occupancy ratios, the two conformations of (–)FTC-PPNP were best modeled with equal occupancies (Supplementary Figure S6). Although similar to (–)3TC-PPNP, the triphosphate moiety in Type-A conformation of (–)FTC-PPNP was bent slightly toward the primer 3′-terminus, allowing a water molecule to slide between Tyr12 and (–)FTC-PPNP (Figure 2C). The conformations adopted by the α- and β-phosphates in the N-shaped conformations of (–)3TC-PPNP and (–)FTC-PPNP are nearly identical to the equivalent α- and β-diphosphate moieties in Dpo4-(–)FTC-DP, Dpo4-(–)3TC-DP and Dpo4-L-dCDP (Figure 3 and Supplementary Figure S7) as well as the ternary structures of Dpo4·DNA·ddNDP (Supplementary Table S3), which display an Λ-shaped diphosphate conformation (Figure 4D). This supports our above conclusion that the (β,γ-imido)-substitution in (–)3TC-PPNP and (–)FTC-PPNP does not significantly affect the binding conformation of an incoming nucleotide. Moreover, it also indicates that either the absence of the 3′-OH of an incoming nucleotide or the mismatching of the nucleotide with a templating base significantly altered the binding conformations of the nucleotide and DNA at the active site of Dpo4. In contrast, the absence of the γ-phosphate moiety did not affect the conformation of an incoming nucleotide and the overall structure of a ternary complex, which was likely due to the positioning of the γ-phosphate moiety on the edge of the Dpo4 surface (Supplementary Figure S8A). Consistently, the nearly superimposable structures of Dpo4-D-dCDP and Dpo4-D-dCTP show that D-dCDP in Dpo4-D-dCDP is bound with an almost identical location and conformation as the counterpart of D-dCTP in Dpo4-D-dCTP (Supplementary Figure S7A).

Bottom Line: Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years.These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180° with an unusual O4'-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase.Such rare binding modes significantly decrease the incorporation rates and efficiencies of these L-nucleotides catalyzed by the polymerase.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.

Show MeSH
Related in: MedlinePlus