title: Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase.
abstract: Considering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. 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. Here we report seven high-resolution ternary crystal structures of a prototype Y-family DNA polymerase, DNA, and D-dCTP, D-dCDP, L-dCDP, or the diphosphates and triphosphates of lamivudine and emtricitabine. 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.
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Vineet
authors.initials: V
authors.lastname: Gaur
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Rajan
authors.initials: R
authors.lastname: Vyas
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Fowler
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Georgia
authors.initials: G
authors.lastname: Efthimiopoulos
authors.affiliation: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94044, USA.
authors.forename: Joy Y
authors.initials: JY
authors.lastname: Feng
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA suo.3@osu.edu.
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000737:chemistry; D003845:Deoxycytosine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D024882:Drug Resistance, Viral; D000068679:Emtricitabine; D007700:Kinetics; D019259:Lamivudine / Q000737:chemistry; D008958:Models, Molecular; D018894:Reverse Transcriptase Inhibitors; D013237:Stereoisomerism; D048229:Sulfolobus solfataricus / Q000201:enzymology
chemical_list: D003845:Deoxycytosine Nucleotides; D018894:Reverse Transcriptase Inhibitors; D003841:Deoxycytidine; C024107:2'-deoxycytidine 5'-triphosphate; D019259:Lamivudine; C045813:2'-deoxycytidine diphosphate; D004247:DNA; D019951:DNA Polymerase beta; D000068679:Emtricitabine
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