Structures of dNTP Intermediate States during DNA Polymerase Active Site Assembly.

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709-2233, USA.
Structure (Impact Factor: 6.79). 09/2012; DOI: 10.1016/j.str.2012.08.008
Source: PubMed

ABSTRACT DNA polymerase and substrate conformational changes are essential for high-fidelity DNA synthesis. Structures of DNA polymerase (pol) β in complex with DNA show the enzyme in an "open" conformation. Subsequent to binding the nucleotide, the polymerase "closes" around the nascent base pair with two metals positioned for chemistry. However, structures of substrate/active site intermediates prior to closure are lacking. By destabilizing the closed complex, we determined unique ternary complex structures of pol β with correct and incorrect incoming nucleotides bound to the open conformation. These structures reveal that Watson-Crick hydrogen bonding is assessed upon initial complex formation. Importantly, nucleotide-bound states representing intermediate metal coordination states occur with active site assembly. The correct, but not incorrect, nucleotide maintains Watson-Crick hydrogen bonds during interconversion of these states. These structures indicate that the triphosphate of the incoming nucleotide undergoes rearrangement prior to closure, providing an opportunity to deter misinsertion and increase fidelity.

  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA/dNTP complexes of DNA polymerase β, several side-chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp192, Arg258, Phe272, Glu295, and Tyr296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants was wild type ≈ R258A > F272A ≈ Y296A > E295A > D192. Since the efficiencies for incorrect insertion was affected to about the same extent for each mutant, effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg258 side-chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. While the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain.
    Journal of Biological Chemistry 09/2014; DOI:10.1074/jbc.M114.607432 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA Polymerases slide on DNA during replication and the interface must be mobile for various conformation changes. The role of lubricant interfacial water has not been understood. In this report, we systematically characterized the water dynamics at the interface and in the active site of a tight-binding polymerase (pol ) in its binary complex and ternary state using tryptophan as a local optical probe. Using femtosecond spectroscopy, we observed that upon DNA recognition the surface hydration water is significantly confined and becomes bound water at the interface, but the dynamics are still ultrafast and occur on the picoseconds time scales. These interfacial water molecules are not trapped but are mobile at the heterogeneous binding nanospace. Combining with our previous observation of ultrafast water motions at the interface of a loose-binding polymerase (Dpo4), we conclude that the binding interface is dynamic and the water molecules in various binding clefts, channels and caves are mobile and even fluid with different levels of mobility for loose or tight binding polymerases. Such a dynamic interface should be general to all DNA polymerase complexes to ensure the biological function of DNA synthesis.
    Biochemistry 08/2014; 53(33). DOI:10.1021/bi500810a · 3.38 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian DNA polymerase (pol) β is the founding member of a large group of DNA polymerases now termed the X-family. DNA polymerase β has been kinetically, structurally, and biologically well characterized and can serve as a phylogenetic reference. Accordingly, we have performed a phylogenetic analysis to understand the relationship between pol β and other members of the X-family of DNA polymerases. The bacterial X-family DNA polymerases, Saccharomyces cerevisiae pol IV, and four mammalian X-family polymerases appear to be directly related. These enzymes originated from an ancient common ancestor characterized in two Bacillus species. Understanding distinct functions for each of the X-family polymerases, evolving from a common bacterial ancestor is of significant interest in light of the specialized roles of these enzymes in DNA metabolism.
    DNA Repair 08/2014; 22C:77-88. DOI:10.1016/j.dnarep.2014.07.003 · 3.36 Impact Factor