Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

Abstract

8-Oxo-7,8,-dihydro-2'-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala(510) and Asn(513), play differential roles in dNTP selectivity. Specifically, Ala(510) and Asn(513) facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.

Figure 1.

Pol λ pre-catalytic complex for 8-oxo-dGTP. (A) The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with 8-oxo-dGTP (magenta) and a 1-nt gapped 16-mer P/T (gray). The upstream primer is terminated with a dideoxynucleotide (ddC) (cyan). (B) 8-Oxo-dGTP (magenta) establishes a Hoogsteen basepair with dA (green). Contoured at 3σ, the simulated annealing F_o – F_c omit density map (gray) for 8-oxo-dGTP is shown. The geometry (C1′ distance and λ angles) of the mismatched base-pair is indicated at the bottom of the figure.

Figure 2.

Pol λ post-catalytic complex for 8-oxo-dGMP incorporation. (A) The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a nicked 16-mer P/T (gray) containing an 8-oxo-dGMP moiety at the primer terminus (magenta). (B) Overlay of the active sites between the pre- and post-catalytic 8-oxo-dGTP/8-oxo-dGMP complexes (RMSD of 0.278 Å over 678 Cα atoms). The β- and ϒ-phosphates that will constitute the pyrophosphate leaving group are highlighted in red.

Figure 3.

Structural features that promote 8-oxo-dGTP incorporation. (A) Ala⁵¹⁰ provides a Van der Waals stacking interaction with the nucleotide of the incoming 8-oxo-dGTP (magenta). (B) Asn⁵¹³ stabilizes 8-oxo-dGTP (magenta) in the syn-conformation. The amino group of the Asn⁵¹³ side chain establishes a hydrogen-bond with the O8 of 8-oxo-dGTP. (C) Comparison of catalytic efficiencies for dTMP incorporation opposite dA using WT, A510D, N513A and A510D/N513A Pol λ. (D) Comparison of catalytic efficiencies for 8-oxo-dGMP(syn) incorporation opposite dA using WT, A510D, N513A and A510D/N513A Pol λ. (E) Comparison of catalytic efficiencies for dGMP incorporation opposite dC using WT, A510D, N513A and A510D/N513A Pol λ. (F) Comparison of catalytic efficiencies for 8-oxo-dGMP(anti) incorporation opposite dC using WT, A510D, N513A and A510D/N513A Pol λ.

Figure 4.

Structural features that discourage incorporation. (A) Comparison of dTTP and 8-oxo-dGTP stacking interactions in the pre-catalytic structures. The stacking interaction between 8-oxo-dGTP(syn) (magenta) and ddC (cyan) at the primer terminus of a complex is shown in the left panel. Comparison with dTTP (magenta) and the ddC (cyan) in the right panel demonstrates that 8-ox-dGTP stacks more poorly (yellow). (B) Overlay of the nascent base-pair in the pre-catalytic 8-oxo-dGTP and dTTP complexes. 8-oxo-dGTP (magenta) appears to displace the templating dA away from its canonical position. The direct consequence of this is that dA shifts further away from Arg⁵¹⁷. The hydrogen-bonding distances for the pre-catalytic 8-oxo-dGTP and dTTP complexes are shown in black and red respectively.