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
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Mentions: Nucleotide selection by DNA polymerases has been hypothesized to be influenced by hydrogen bonds between a nascent base pair (1,2), base stacking (3), minor groove hydrogen bonds with protein residues (4,5), nucleotide desolvation (6), induced-fit protein conformational change (7–12), phosphodiester bond formation (13), positive and negative selectivity (14) and steric repulsion (15,16). In addition, shape complementarity (or geometric selection) has also been proposed to influence polymerase fidelity based on different tightness of the active sites of high- and low-fidelity DNA polymerases and different overall shapes of correct and incorrect base pairs (3). If geometric selection is stringent, it will be unlikely for a DNA polymerase to incorporate a nucleotide analog with L-stereochemistry (L-dNTP) opposite a natural templating nucleotide with D-stereochemistry. However, because many DNA polymerases and reverse transcriptases (RTs) can incorporate non-physiological nucleotide analogs, it might be possible for a polymerase to relax its D-stereoselectivity to bind and incorporate L-dNTPs or their analogs. This is more likely when considering that low-fidelity DNA polymerases and RTs possess flexible active sites and lack a proof-reading 3′ → 5′ exonuclease activity. The D-stereoselectivity of DNA polymerases and RTs has been further shown to be relaxed based on the successful development of two nucleoside reverse transcriptase inhibitors (NRTIs): lamivudine ((–)3TC; (–)-β-L-2′-3-dideoxy-3′-thiacytidine) and its 5-fluorinated derivative, emtricitabine ((–)FTC; (–)-β-L-2′-3-dideoxy-5-fluoro-3′-thiacytidine) (Figure 1), which terminate genomic replication of human immunodeficiency viruses (HIV) once converted to their triphosphate forms ((–)3TC-TP), (–)FTC-TP) and subsequently incorporated by HIV RT in vivo. Both (–)3TC and (–)FTC have been shown to be clinically more effective and less toxic than their enantiomeric D-isomers (17–21). Furthermore, (–)3TC and the third L-drug telbivudine (L-thymidine) have been used as drugs for the treatment of hepatitis B virus (HBV) infections (22,23). Like other NRTIs, the three L-drugs (lamivudine, emtricitabine and telbivudine) NRTIs including (–)3TC and (–)FTC also cause clinical side effects and some of them are likely associated with the inhibition of human DNA polymerases (16). Among 16 identified human DNA polymerases in the A-, B-, X- and Y-families, our recent kinetic analysis demonstrates that the DNA damage repair X-family (β and λ) and the lesion-bypass Y-family polymerases (η, κ, ι and Rev1) are more prone to inhibition by the triphosphates of the L-drugs than the A- and B-family, replicative polymerases (24).
Affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.