Substitution of Ala for Tyr567 in RB69 DNA polymerase allows dAMP and dGMP to be inserted opposite Guanidinohydantoin

Abstract

Continuous oxidative damage inflicted on DNA produces 7,8-dihydro-8-oxoguanine (8-oxoG), a commonly occurring lesion that can potentially cause cancer by producing G → T transversions during DNA replication. Mild oxidation of 8-oxoG leads to the formation of hydantoins, specifically guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), which are 100% mutagenic because they encode almost exclusively the insertion of dAMP and dGMP (encoding G → T and G → C transversions, respectively). The wild-type (wt) pol α family DNA polymerase from bacteriophage RB69 (RB69pol) inserts dAMP and dGMP with low efficiency when situated opposite Gh. In contrast, the RB69pol Y567A mutant inserts both of these dNMPs opposite Gh with >100-fold higher efficiency than wt. We now report the crystal structure of the "closed" preinsertion complex for the Y567A mutant with dATP opposite a templating Gh (R-configuration) in a 13/18mer primer-template (P/T) at 2.0 Å resolution. The structure data reveal that the Y to A substitution provides the nascent base pair binding pocket (NBP) with the flexibility to accommodate Gh by allowing G568 to move in the major-to-minor groove direction of the P/T. Thus, Gh is rejected as a templating base by wt RB69pol because G568 is inflexible, preventing Gh from pairing with the incoming dATP or dGTP base.

Figure 1

8-oxoG compared with its oxidized products Gh and Sp. A. Structures of an incoming dCTP or dATP paired opposite a templating 8-oxoG. B. Hydantoin products of one-electron oxidation of 8-oxoG, Gh and Sp, via a 5-OH-8-oxoG intermediate. C. Slight rotation of Gh relative to the 2′-deoxyribose sugar (indicated with a curved arrow) forms a base-pair with dATP with Gh in a “high-syn” conformation. Note how the base-pair “buckles” (the arrows pointing away from the Gh sugar indicate that the backbone continues in the 5′ and 3′ directions). D. Numbering scheme of Gh, shown in the R-configuration at C-4.

Figure 2

Steric relationship of some of the NBP residues surrounding an incoming dTTP paired opposite a templating dA (from ref (34), PDB entry 1IG9). The nascent base-pair and surrounding residues are shown from the duplex side of the primer-template in space-filling form. The nascent base-pair is shown in blue, and the terminal C:G base-pair, with C located at the primer-terminus, is shown in stick form (purple) for the sake of clarity. L561 and Y567 are colored orange.

Figure 3

Kinetics of dNMP insertion by wt RB69pol and the Y567A mutant. A. Insertion of dNMPs opposite Gh and 8-oxoG by wt RB69pol. P represents the primer, and P+1 represents the primer extended by one nucleotide. Oxidation of 8-oxoG → Gh results in dramatically reduced incorporation of dCMP, to the point that it cannot be observed. B. Kinetics of insertion of dAMP opposite Gh by wt. Progress curves at various [dATP]s fit to single-exponential equations. C. Plot of k_obs vs [dATP] fit to eq 2. D. Kinetics of insertion of dAMP opposite Gh by the Y567A mutant. Same as panel B but with data obtained using the Y567A mutant. E. Same as panel C using the results in panel D.

Figure 4

Progress curves showing the rates of bypass of thymine glycol (Tg) and 1,3-diaza-2-oxophenoxazine (tC^o) by wt RB69pol and the Y567A mutant. For reference, P/T sequences and structures of the analogs are shown. Progress curves for insertion of dNMPs were obtained under single turnover conditions without trap DNA, and with 1 mM dNTPs (see Experimental Procedures). A. Rate of DNA product formation for insertion of dAMP and dGMP opposite Tg, and the rate of extension past A:Tg and G:Tg base-pairs (via insertion of dTMP, the next correct dNMP), by wt RB69pol (●) and the Y567A mutant (▲). B. Rate of DNA product formation for insertion of dAMP and dGMP opposite tC^o, and extension past A:tC^o and G:tC^o base-pairs, by wt RB69pol (●) and the Y567A mutant (▲). * After oligonucleotide synthesis and deprotection, Tg is predominantly in the cis-5R,6S configuration (40).

Figure 5

Crystal structure of the RB69pol Y567A mutant in a complex with a dATP:Gh base-pair and its relationship to residues in the NBP. A. Omitted Fo – Fc electron density map of the dATP:Gh nascent base-pair. Note the number of water molecules that surround the guanidinium group, and the water-mediated hydrogen bonding network that links Gh and dATP (pointed to with an arrow). B. Comparison of the crystal structures of wt and the Y567A mutant in a complex with dCTP:dG and dATP:Gh base-pairs, respectively. Superimposed palm domains show that the Y567A substitution resulted in a shift of G568 into the DNA minor-groove by ~0.8 Å. The wt:dCTP:dG backbone and base-pair is colored yellow, and the Y567A:dATP:Gh backbone is colored orange. The dCTP:dG base-pair is shown in yellow, and the dATP:Gh base-pair is shown with dATP colored orange, and Gh colored blue.

Figure 6

Possible “wobble” configurations of the dTTP:dG and dATP:Gh mispairs in the Y567A mutant complex. A. Downward shift of dG into the DNA minor groove on the templating side (against G568) leads to a “wobble” base-pair between dG and dTTP. B. Downward shift of dGTP into the DNA minor groove into the space provided by the Y567 to A substitution leads to an “inverted wobble” base-pair between dGTP and Gh.

Figure 7

In silico modeling of dAMP:Gh as a terminal (n-1) base-pair and its relationship to surrounding amino acid side-chains in the Y567A mutant. A. The terminal base-pair ddC:dG in the Y567A:dATP:Gh structure (orange) and the hydrogen bonding network it forms between the 5′-bridging phosphate of dG and side-chains N572 and W574. B. Superposition of the glycosidic bonds of the dATP:Gh base-pair onto the ddC:dG base-pair. Black arrows denote unfavorable interactions. C. Superposition of the dATP:syn-8-oxoG base-pair onto the ddC:dG base-pair. Black arrows denote unfavorable interactions (29). Note that both Gh and syn-8-oxoG form unfavorable contacts with their 5′-terminal phosphates.