Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7

Abstract

Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg²⁺, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg²⁺ to an active site because Mg²⁺ is spectroscopically silent and Mg²⁺ binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg²⁺ with Mn²⁺:Mn²⁺ that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn²⁺ is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn²⁺ that is free in solution and Mn²⁺ bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

Keywords: DNA polymerase; XAFS; manganese; metal cofactor; metalloenzyme.

Figure 1

(Color online) Crystal structure of the bacteriophage T7 DNA polymerase (PDB entry 1t8e). The DNA polymerase (gp5) and the host encoded processivity factor trx are indicated. The figure was created using PyMOL (http://www.pymol.org).

Figure 2

(Color online) Schematic representation of sample preparation. Bound nucleotide obtained upon titration of T7 DNA polymerase (gp5) above and below its binding constant to dATP (20 µM) using the binding equation (1). The four samples are depicted schematically in columns where black is the amount of DNA polymerase used, gray is the amount of trx, and the amount of Mn-ddATP is fixed as indicated (300 µM).

Figure 3

Effect of substitution of Mg with Mn on T7 DNA polymerase activity. DNA polymerase activity was measured in a reaction containing 40 mM tris-HCl, pH 7.5, 2.75 mM MgCl2, 10 mM DTT, 50 mM potassium glutamate, 0.25 mM dTTP, dCTP, dGTP, and [α-32P] dTTP, 20 nM primed M13 DNA, and 10 nM T7 gp5/trx. After incubation at 37 °C for 10 min, the amount of [α³²-P] dTMP incorporated into DNA was measured.

Figure 4

(Color online) (a) Site directed titration XANES analysis of T7 DNA polymerase above and below its binding constant to Mn-dNTP [according to Eq. (1), experimental]. Each sample contained the same concentration of Mn-dNTP (300 µM) and increasing concentrations of T7 DNA polymerase (blue: 0 µM; light blue: 32-µM; orange; 170-µM; red: 450-µM) mixed with E. coli thioredoxin (450-µM) (see Figure 2). (b) A typical binding curve when each point in this curve represents the maximum amplitude value of each XANES spectrum vs. dNTP binding [color coding as in (a)].