Kinetics of mismatch formation opposite lesions by the replicative DNA polymerase from bacteriophage RB69

Abstract

The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k(pol)) were generally higher for the variant, the presence of a lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG.dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.

Figure 1

The lesions studied in this work. Top: The oxidation of 2′-deoxyguanosine leads to 8-oxo-7,8-dihydro-2′-deoxyguanosine. Middle: The oxidation of 2′-deoxyadenosine generates 2-hydroxy-2′-deoxyadenosine. Bottom: A naturally occurring abasic site (hemiacetal form) (left) and a non-hydrolyzable form, tetrahydrofuran (F) (right).

Figure 2

Representative kinetic data. The product formation curve for incorporating 200 μM dTTP opposite 2-OHA by the L561A variant (A) was fit to a double exponential equation (P=A(1-e^−k_fast*t)+B(1-e^−k_slow*t)) and for incorporation of 200 μM dATP opposite F (C) was fit to a single exponential equation (P=A(1-e^−k*t)). The rate constants for each reaction were plotted against dNTP concentrations and fit to a square hyperbola (k_obs = (k_pol [dNTP])/(K_D,APP [dNTP])) for 2-OHA•dTMP (B) and F•dAMP (D).

Figure 3

The 8-oxoG•dATP mispair in the active site of the RB69 DNA polymerase. An Fo-Fc map is shown (green; contoured at 2.5 σ), along with an anomalous difference Fourier map (orange, contoured at 4.5 σ) pinpointing the location of the two manganese ions (purple spheres). dATP fits the density in a normal anti conformation while the electron density shows clear evidence that 8-oxoG adopts a syn conformation. The primer strand of DNA has been omitted to aid in clarity.

Figure 4

Comparison of 8-oxoG•dNTP ternary complexes. In these images, the structures have been aligned using the α-carbons of the palm domain (residues 383-468 and 573-729). The duplex DNA from all structures was omitted for the sake of clarity. (A) The ternary complex with a 8-oxoG (anti)•dCTP base pair (PDB ID: 1Q9Y) (9) is shown as a cartoon (tan) and the nascent base pair is shown in magenta. Van der Waals surfaces are shown for the residues forming the nucleotide binding pocket (yellow), 8-oxoG (gray) and L561 (light green). (B) An orthogonal view to (A) illustrating the proximity between 8-oxoG (anti) and L561. (C) The ternary complex of 8-oxoG (syn)•dATP from this work is shown in pale green and the nascent base pair in cyan. (D) An orthogonal view to (C) predicting an unfavorable contact between 8-oxoG (syn) and A561. In this view a leucine residue (pale gray) has been modeled onto the alanine at position 561 to show the unfavorable interaction that may develop between 8-oxoG (syn) and L561.

Figure 5

A cavity is exposed when leucine 561 is substituted with alanine. (A) The polymerase domain of the current work is shown as a pale green surface, a modeled leucine 561 is shown in magenta and serine 565 is shown in tan. The leucine and serine residues block the cavity in this image. (B) Removal of the leucine residue opens up this cavity and may provide an opportunity for alternate template configurations to occur during DNA synthesis. (C) Mutation of serine 565 to glycine as described previously (52) shows an even more exposed cavity.