Abstract
The 2,4-difluorotoluene (DFT) analog of thymine has been used extensively to probe the relative importance of shape and hydrogen bonding for correct nucleotide insertion by DNA polymerases. As far as high fidelity (A-class) polymerases are concerned, shape is considered by some as key to incorporation of A(T) opposite T(A) and G(C) opposite C(G). We have carried out a detailed kinetic analysis of in vitro primer extension opposite DFT-containing templates by the trans-lesion (Y-class) DNA polymerase Dpo4 from Sulfolobus solfataricus. Although full-length product formation was observed, steady-state kinetic data show that dATP insertion opposite DFT is greatly inhibited relative to insertion opposite T (approximately 5,000-fold). No products were observed in the pre-steady-state. Furthermore, it is noteworthy that Dpo4 strongly prefers dATP opposite DFT over dGTP (approximately 200-fold) and that the polymerase is able to extend an A:DFT but not a G:DFT pair. We present crystal structures of Dpo4 in complex with DNA duplexes containing the DFT analog, the first for any DNA polymerase. In the structures, template-DFT is either positioned opposite primer-A or -G at the -1 site or is unopposed by a primer base and followed by a dGTP:A mismatch pair at the active site, representative of a -1 frameshift. The three structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite DFT and its inability to extend beyond a G:DFT pair. Although hydrogen bonding is clearly important for error-free replication by this Y-class DNA polymerase, our work demonstrates that Dpo4 also relies on shape and electrostatics to distinguish between correct and incorrect incoming nucleotide.
Highlights
Bic T isostere 2,4-difluorotoluene (DFT)3 (Fig. 1) and other analogs with substituents of increasing size at the 2- and 4-positions of the aromatic moiety appear to support different mechanisms of nucleotide insertion by high fidelity (A-class) and trans-lesion (Y-class) DNA polymerases
A-class DNA polymerases whose activities were assessed using apolar T analogs to date include Escherichia coli pol I [3,4,5] and the polymerase from phage T7 [6], and the tested Y-class DNA polymerases consisting of yeast pol [7], human pol [8], and the Dbh (DinB homolog [5]) and Dpo4 [9] polymerases from Sulfolobus acidocaldarius and Sulfolobus solfataricus, respectively
As far as the different roles of sterics versus hydrogen bonding in the replications catalyzed by A-class and Y-class DNA polymerases are concerned, recent crystal structures of a the high fidelity Bacillus stearothermophilus DNA polymerase I large fragment (BF) and Dpo4 in complex with DNAs containing the O6-methyl-G (O6-MeG) adduct opposite C and T may be instructive
Summary
Materials—Dpo was expressed in E. coli and purified to electrophoretic homogeneity as described previously [20]. LC-MS/MS Analysis of Oligonucleotide Products from Dpo Reactions—Dpo (5 M) was preincubated with primer-template DNA (10 M), and the reaction was initiated by addition of dNTP (1 mM each) and MgCl2 (5 mM) in a final volume of 100 l. Dpo Pyrophosphorolysis Activity—The effect of DFT upon the pyrophosphorolysis activity of Dpo was tested by incubating Dpo (100 nM) with a radiolabeled DNA substrate (100 nM) in 50 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl, 10.0 mM DTT, 200 g mlϪ1 BSA, and 2.5% glycerol (v/v) at 60 °C. In the case of the Dpo4(DFT:13A) complex crystals were obtained by equilibrating droplets against reservoir solution containing 12% polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M calcium acetate, and 20 mM Tris (pH 7.5). The crystallographic figures were prepared with the program PyMOL [43]
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