Substitution of Ala for Tyr567 in RB69 DNA polymerase allows dAMP to be inserted opposite 7,8-dihydro-8-oxoguanine

Abstract

Accurate copying of the genome by DNA polymerases is challenging due in part to the continuous damage inflicted on DNA, which results from its contact with reactive oxygen species (ROS), producing lesions such as 7,8-dihydro-8-oxoguanine (8-oxoG). The deleterious effects of 8-oxoG can be attributed to its dual coding potential that leads to G --> T transversions. The wild-type (wt) pol alpha family DNA polymerase from bacteriophage RB69 (RB69pol) prefers to insert dCMP as opposed to dAMP when situated opposite 8-oxoG by >2 orders of magnitude as demonstrated using pre-steady-state kinetics (k(pol)/K(d,app)). In contrast, the Y567A mutant of RB69pol inserts both dCMP and dAMP opposite 8-oxoG rapidly and with equal efficiency. We have determined the structures of preinsertion complexes for the Y567A mutant with dATP and dCTP opposite a templating 8-oxoG in a 13/18mer primer-template (P/T) at resolutions of 2.3 and 2.1 A, respectively. Our structures show that the 8-oxoG residue is in the anti conformation when paired opposite dCTP, but it flips to a syn conformation forming a Hoogstein base pair with an incoming dATP. Although the Y567A substitution does not significantly change the volume of the pocket occupied by anti-8-oxoG, it does provide residue G568 the flexibility to move deeper into the minor groove of the P/T to accommodate, and stabilize, syn-8-oxoG. These results support the hypothesis that it is the flexibility of the nascent base pair binding pocket (NBP) in the Y567A mutant that allows efficient insertion of dAMP opposite 8-oxoG.

Figure 1

Structures of an incoming dCTP or dATP paired opposite a templating 8-oxoG. 8-OxoG likely flips into the syn conformation to pair opposite dATP to form a base pair comparable to dATP:T, with two inter-base hydrogen bonds.

Figure 2

Steric relationship of some of the NBP residues surrounding an incoming dTTP paired opposite a templating A (from Franklin et al., PDB code 1IG9 (20)). A. The nascent base-pair and surrounding residues are shown from the duplex side of the primer-template in space-filling form (the terminal base-pair is shown in stick form (purple) for clarity). L561 and Y567 are shown in orange. B. Orthogonal view of (A), showing the close proximity of L561 with the templating base.

Figure 3

Kinetics of dAMP insertion opposite 8-oxoG by the Y567A mutant (A and B) and the wt (C and D). A. Progress curves with various [dATP], 10, 40, 100, 250, 700, and 1500 µM (bottom to top), fit to single exponential equations. 1 mg/ml Heparin was used to prevent rebinding of the enzyme to the P/T. B. Plot of k_obs versus [dATP] fit to Equation 2 to obtain k_pol and K_d,app. C. Same as A but with the wt. Progress curves were obtained without a heparin trap. D. Same as B using the results of C.

Figure 4

Purine:purine nascent base pairs poorly utilized by the Y567A mutant.

Figure 5

Progress curves for wt RB69pol and the Y567A mutant for insertion of d(3DA)MP opposite T and dTMP opposite 3DA. A. Shown is the purine numbering scheme (left) and 3-deazaadenine (3DA) (right). B. Rate of DNA product formation for dTMP insertion opposite 3DA by wt (▲), dTMP opposite A by wt (▼), and dTMP opposite 3DA by the Y567A mutant (■). C. Rate of DNA product formation for d(3DA)TP insertion opposite T by wt (■), dATP opposite T by wt (●), and dTMP opposite 3DA by the Y567A mutant ▲.

Figure 6

k_obs versus [dTTP] plots of wt RB69pol extending primers beyond A:8-oxoG (●) and C:8-oxoG (▼) terminal base-pairs.

Figure 7

Crystal structures of the RB69pol Y567A mutant in complex with anti-8-oxoG:dCTP and syn-8-oxoG:dATP. A. Fo-Fc electron density map of the anti-8-oxoG:dCTP nascent base-pair. B. Fo-Fc electron density map of the syn-8-oxoG:dATP nascent base-pair.

Figure 8

Comparison of the crystal structures of wt and the Y567A mutant in complex with anti-8-oxoG:dCTP (14). A. Superimposed palm domains showing that the Y567A substitution resulted in a shift of G568 by ~ 0.7 Å laterally toward Y416, relative to wt. Residues of the Y567A mutant and wt are shown in yellow and orange, respectively, and the individual residues Y567 and Y567A are shown in gray and magenta, respectively. The nascent base-pair in the wt and Y567A mutant structures are shown in silver and blue, respectively. B. Schematic illustration of the spatial relationship between G568 and the nascent base-pair as shown in (A).

Figure 9

Comparison of the Y567A mutant anti-8-oxoG:dCTP and syn-8-oxoG:dATP ternary crystal structures. A. Superimposed palm domains showing that 8-oxoG flips from anti to a syn conformation resulting in a shift of A567 and G568 into the minor groove of the primer-template by ~ 0.6 Å. The Y567A:anti-8-oxoG:dCTP backbone is shown in orange, and the Y567A:syn-8-oxoG:dATP backbone in yellow. B. Schematic illustration of the spatial interaction between G568 and the nascent base-pair as shown in (A).

Figure 10

Computer modeling of wt and the Y567A mutant ternary complex with the observed dATP:8-oxoG or dCTP:8oxoG translocating from the nascent base-pairing position to the product position. A. Structure showing the terminal template base G (dG(N-1)) opposite the dideoxy-terminated nucleotide dC in the wt RB69pol ternary complex structure with Y567A modeled into the structure (PDB entry 1IG9). The mutation site is shown in magenta. Hydrogen bonds are shown in green dotted lines. B. Corresponding wt structure showing that Y567 and Y391 can form a hydrogen bond. C. Superposition of the anti-8-oxoG:dCTP base-pair onto the dG(N-1):dC base-pair. Black arrows illustrate which interactions are unfavorable. D. Superposition of the syn-8-oxoG:dATP base-pair onto the dG(N-1):dC base-pair. Black arrows illustrate which interactions are unfavorable.