Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase.
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.
Affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.Show MeSH
Related in: MedlinePlus
Mentions: After numerous attempts, we successfully crystallized ternary complexes of Dpo4, a DNA substrate 13/18-mer (see the ‘Materials and Methods’ section), and either D-dCTP, L-dCTP, (–)3TC-TP or (–)FTC-TP. L-dCTP and its two analogs were found in their diphosphate forms (Figure 2E and F and Supplementary Figure S3), which was caused by the weak phosphatase activity of Dpo4 (38). In contrast, D-dCTP was intact with its triphosphate moiety adopting a chair-like conformation (Figure 2A). To capture the γ-phosphate of the two L-NRTIs in crystal structures, non-hydrolyzable (–)3TC-PPNP and (–)FTC-PPNP (Figure 1) were synthesized and used in subsequent crystallization trials. Although slowly, both (–)3TC-PPNP and (–)FTC-PPNP can be incorporated into the 13/18-mer by Dpo4 with rates of 1.1 × 10−4 and 1.5 × 10−4 s−1, respectively (Supplementary Figure S2M–O). Notably, it has been demonstrated that substitutions of β,γ-oxygen result in very low nucleotide incorporation efficiencies by DNA polymerases but have no effect on the binding conformation of these nucleotide analogs in the ternary crystal structures (45). Together, these results suggest that (–)3TC-PPNP and (–)FTC-PPNP can be good models for (–)3TC-TP and (–)FTC-TP, respectively, and the conformations of these β,γ-substituted nucleotide observed in crystal structures will closely resemble the possible conformations adopted by (–)3TC-TP and (–)FTC-TP within the active site. The crystal structures of seven Dpo4·DNA·nucleotide ternary complexes were refined to resolutions of 1.8–2.4 Å and are referred to as Dpo4-D-dCTP, Dpo4-D-dCDP, Dpo4-L-dCDP, Dpo4-(–)FTC-DP, Dpo4-(–)3TC-DP, Dpo4-(–)FTC-PPNP and Dpo4-(–)3TC-PPNP (Table 2, Supplementary Figures S3–S5). Notably, all seven complexes were crystallized in orthorhombic space group. Six of them were crystallized in P21212 space group with one ternary complex molecule in an asymmetric unit while Dpo4-L-dCDP was crystallized in P212121 space group with two ternary complex molecules (chains A and D for Dpo4) in an asymmetric unit (Supplementary Figure S3). Notably, the overall structures of Dpo4 in the seven complexes are almost identical with root-mean-square deviations between 0.37 and 0.51 Å (Supplementary Table S1, Supplementary Figures S3–S5). Thus, the overall structure of Dpo4 was not significantly affected by either the binding of a nucleotide with L-stereochemistry, the absence of the γ-phosphate in the incoming nucleotide, or the lack of primer 3′-OH group.
Affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.