Substrate-Dependent Millisecond Domain Motions in DNA Polymerase beta

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Journal of Molecular Biology (Impact Factor: 3.96). 03/2012; 419(3-4):171-82. DOI: 10.1016/j.jmb.2012.03.013
Source: PubMed

ABSTRACT DNA polymerase β (Pol β) is a 39-kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers, and these mutations are further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function, and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate-bound, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s(-1). Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped DNA-substrate-bound form. Upon binding DNA, these lyase domain motions are significantly quenched, whereas evidence for conformational motions in the polymerase domain becomes apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity.

  • [Show abstract] [Hide abstract]
    ABSTRACT: We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by co-localization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average life-time of this state by 140% relative to undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes.
    Nano Letters 09/2014; 14(10). DOI:10.1021/nl502890g · 12.94 Impact Factor
  • [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 · 3.19 Impact Factor

Rebecca Beth Berlow