Lesion (in)tolerance reveals insights into DNA replication fidelity

Abstract

The initial encounter of an unrepaired DNA lesion is likely to be with a replicative DNA polymerase, and the outcome of this event determines whether an error-prone or error-free damage avoidance pathway is taken. To understand the atomic details of this critical encounter, we have determined the crystal structures of the pol alpha family RB69 DNA polymerase with DNA containing the two most prevalent, spontaneously generated premutagenic lesions, an abasic site and 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG). Identification of the interactions between these damaged nucleotides and the active site provides insight into the capacity of the polymerase to incorporate a base opposite the lesion. A novel open, catalytically inactive conformation of the DNA polymerase has been identified in the complex with a primed abasic site template. This structure provides the first molecular characterization of the DNA synthesis barrier caused by an abasic site and suggests a general mechanism for polymerase fidelity. In contrast, the structure of the ternary 8-oxodG:dCTP complex is almost identical to the replicating complex containing unmodified DNA, explaining the relative ease and fidelity by which this lesion is bypassed.

Figure 1

Oligonucleotide sequences and DNA adducts. (A) The 14 nt primers are identical in all trials and terminated by ddC (indicated by C^*). Template strands are 18 nt long with a 3′-dG overhang. X denotes the position of the lesion for primer/template combinations (1) and (2), and the arrow the position of dNTP incorporation. (B) Structures of the lesions at position X in the templates 8-oxodG and tetrahydrofuran (abasic site model).

Figure 2

Structure of RB69 pol/DNA complexes. (A) Overall structure of the replicating 8-oxodG:dCTP complex formed between RB69 pol, DNA sequence 1 (Figure 1A, X=8-oxodG), and dCTP. RB69 pol is colour coded according to its five domains (N-terminal domain: residues 1–108 and 340–382 in yellow; 3′–5′ exonuclease domain: residues 109–339 in red; palm domain: residues 383–468 and 573–729 in magenta; fingers domain: residues 469–572 in blue; and thumb domain: residues 730–903 in green) as well as the primer (dark green) and template (dark magenta) DNA strands and the dCTP (dark green). (B) Final SIGMAA-weighted 2F_o−F_c electron density map contoured at 1σ, covering the 8-oxodG:dCTP base pair as well as the four base pairs preceding the nascent base pair. Ca²⁺ ions are depicted as yellow spheres. (C) Top view of the nascent 8-oxodG:dCTP base pair with the SIGMAA-weighted 2F_o−F_c electron density contoured at 1σ obtained after molecular replacement with the dA:dTTP-containing structure. Difference electron density at 3σ is shown in red and marks the positions of the O8 and N2 groups of 8-oxodG. (D) Overall structure of the AP:dG complex (molecule C) formed by RB69 pol, DNA sequence 1 (Figure 1A, X=AP, including an additional G at the template 3′-end) and dGMP, colour coded as in (A). (E) Electron density map of the DNA (analogous to (B)). (F) Top view of the AP:dGMP ‘pair' featuring the final electron density map contoured at 1σ. Figures 2, 3, 4, 5, 6 and 7 were generated with Molscript (Kraulis, 1991) and Raster3D (Merrit and Murphy, 1994).

Figure 3

The active site. (A) Superposition of the 8-oxodG:dCTP base pair (stick mode) with the dA:dTTP base pair (CPK mode) of the replicating complex with unmodified DNA. Also shown are the amino acids of the active site (red: highly conserved, green: type conserved, black: not conserved). (B) Polymerase active site with the nascent AP:dGMP ‘pair'. (C) Superposition of active site amino acids of the 8-oxodG:dCTP complex (colour coded according to the different domains as in Figure 2A) and the AP:dG complex (in light gray). Numbers denote displacements of Cα main chain atoms (for a complete list, see supplementary data, Supplementary Table S1). The dA:dTTP base pair has been included as in part A.

Figure 4

Comparison of the AP:dG and replicative complexes. (A) Ribbon presentation of the AP:dG complex, colour coded with respect to rms deviations from the replicating complex. The superposition is based on the Cα atoms of the palm domain (residues 383–468, 573–729), as they define a relatively rigid unit of the protein. Rms deviations for the Cα atoms of the other four domains were calculated using RMSPDB (Kleywegt et al, 2001). Gray colour denotes virtually no changes, while red colour shows maximum domain shifts (see depicted colour code). (B) Detail of (A), showing the fingers, palm, and N-terminal domains of the AP:dG complex and additionally the fingers domain of the replicative complex (dark gray) to illustrate the domain shift. The DNA is shown in green/magenta for the AP:dG complex and yellow for the replicating complex.

Figure 5

Intermolecular interactions in the AP:dG complex. (A) Contacts between neighboring molecules in the AP:dG complex. The 3′-dG of each template strand is bound to a specific binding pocket in the N-terminal domain of an adjacent polymerase molecule. (B) Final SIGMAA-weighted 2F_o−F_c electron density map of the binding pocket at 1σ for the amino acids, part of the template (magenta) as well as the primer strand (green) revealing the presence of an additional unpaired dG at the 3′-end of the template. (C) Terminal transferase activity of RB69 pol in the presence of dideoxy-terminated primed templates (P:T) containing an abasic site (AP) or 8-oxodG. Primer and template bands are indicated with arrows. P:T was incubated with RB69 pol in the absence (lane 1) or presence of dGTP (lanes 2 and 6), dATP (lane 3), dCTP (lane 4), or dTTP (lane 5).

Figure 6

Modeling of base pairs in the polymerase active site. (A) Model of an 8-oxodG:dATP base pair in the polymerase active site of RB69 pol. The syn conformation of 8-oxodG (green) leads to steric interference between O8 and the Cα atom of Gly 568. (B) Model of an 8-oxodG:dA base pair poised for extension. Modeling the 8-oxodG base in the syn conformation generates only a contact (yellow arrow) to a water molecule. (C) Model of an 8-oxodG:dC base pair poised for extension. The anti conformation of 8-oxodG leads to a steric clash with the phosphate group. (D) Nucleotide selection during insertion opposite 8-oxodG. RB69 pol (0–19 nM) was incubated with P:T (Figure 1A, sequence 1) in the presence of 5 μM dCTP (closed circles) or dATP (open circles), as described in Materials and methods. The % of extended primer was plotted versus the RB69 pol concentration. (E) Extension of an 8-oxodG:dC (closed circles) or an 8-oxodG:dA (open circles) base pair as described above, but using 5 μM dTTP instead of dCTP.

Figure 7

Influence of Gly 568 on DNA binding to the polymerase active site. Column (I) shows the event of nucleotide insertion opposite an unmodified template strand with adenine in the active site, while column (II) depicts the case of a template containing an abasic site as in the AP:dG complex. Vertical arrows specify the strained (red) or the relaxed state (green), respectively. Diagonal arrows indicate whether the polymerase is in the closed (red) or open conformation (green). The template strand is depicted in magenta and the incoming nucleotide in green. The yellow box indicates the position of Gly 568. (IA) and (IIA) show the polymerase in the strained state and the open conformation. Transition into the relaxed state presumably causes the adenine base of the unmodified template to be pushed back (IB), while the AP-containing template is unaffected (IIB). (IC) and (IIC) depict an incoming dNTP bound to the base of the fingers domains. Transition to the closed and strained conformation ensures the correct positioning of all residues to enable the catalytic phosphodiester bond formation (ID). In the case of AP, a closed and relaxed conformation is feasible (IID). The missing complementary base causes the dNTP to be held in place less tightly and phosphodiester bond formation is less efficient (IIE).