DNA Polymerase β. 5. Dissecting the Functional Roles of the Two Metal Ions with Cr(III)dTTP1

Abstract

DNA polymerases catalyze the synthesis of DNA with a fidelity ranging from 1000 to 100 000.2 Although the kinetic properties of DNA polymerases have been investigated extensively,3-5 the physical origin of this high fidelity is still poorly understood. The crystal structures of DNA polymerase â (Pol â) from rat brain show the presence of two Mg2+ ions in the active site6a,b,7 (Figure 1). Functionally, it has been established that DNA polymerases require divalent metal ions for catalysis.8,9 However, no kinetic evidence that two divalent metal ions are required has been obtained for DNA polymerases, nor have the roles of the two metal ions been dissected functionally. We here report the use of Cr(III)dNTP coupled with stopped-flow fluorescence to show the requirement of two metal ions, and to dissect their functional roles in the catalysis by Pol â from rat brain (overexpressed in Escherichia coli).5 Pol â can serve as a good model for nucleotidyl transfer reactions because of its small size (39 kDa) and simplicity (without exonuclease or proof-reading activities). Using a synthetic DNA primer/template containing the 2-aminopurine nucleotide analogue (2-AP) (Figure 2A), we recently reported two phases of fluorescence changes in the stopped-flow fluorescence assay of nucleotide incorporation catalyzed by Pol â (Figure 2B).10 The fast phase was attributed to a conformational change step because its rate dependence on [dTTP] is hyperbolic. The rate of the slow phase corresponds to product formation. With a dideoxynucleotide-terminated primer, we have successfully dissected the slow fluorescence change and the chemical step, and attributed the slow phase to a conformational change that is the rate-limiting step in the reaction.10 Cr(III)‚nucleotide complexes have been extensively used as analogues of Mg(II)‚nucleotide complexes to solve enzymological problems because of their exchange-inert properties.11-13 When the Pol â‚DNA complex was mixed with â,γ-bidentate Cr(ΙΙΙ)dTTP14 in the absence of Mg2+, only the fast phase was observed (Figure 2C). However, the slow phase was restored upon addition of Mg2+ (Figure 2D). Under the latter condition the substrates were turned over, and the rate of the slow phase was comparable to the rate of product (21-mer) formation measured by the rapid chemical quench assay. In the absence of any metal ions (dTTP alone) neither fluorescence changes nor catalysis occurred. These results clearly indicate that the fast conformational change is induced by the binding of Cr(III)dTTP and the slow conformational change by the binding of Mg2+. On the basis of the crystal structure shown in Figure 1, Cr3+ and Mg2+ should occupy the metal ion sites A and B, respectively.6c The results described above, taken together with our pre-steadystate kinetic analyses reported previously, strongly support the mechanism we proposed for Pol â:10