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. Author manuscript; available in PMC: 2011 Nov 10.
Published in final edited form as: Structure. 2010 Nov 10;18(11):1463–1470. doi: 10.1016/j.str.2010.08.019

Structural basis for error-free replication of oxidatively damaged DNA by yeast DNA polymerase η

Timothy D Silverstein 1, Rinku Jain 1, Robert E Johnson 2, Louise Prakash 2, Satya Prakash 2, Aneel K Aggarwal 1,*
PMCID: PMC3044447  NIHMSID: NIHMS245470  PMID: 21070945

Summary

7,8-dihydro-8-oxoxguanine (8-oxoG) adducts are formed frequently by the attack of oxygen FREE radicals on DNA. They are amongst the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase η (Polη). We show that the open active site cleft of Polη can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and postinsertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Polη to prevent the mutagenic replication of 8-oxoG lesions in cells.

Introduction

Reactive oxygen species are a major source of DNA damage (Ames et al., 1993). Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoxguanine (8-oxoG) is one of the most common lesions formed (Beckman and Ames, 1997; Helbock et al., 1998). The presence of 8-oxoG in cells has been implicated in number of diseases including cancers, neurodegenerative and cardiovascular disorders, as well as ageing (Ames et al., 1993; Cooke et al., 2003; Lu et al., 2004). 8-oxoG is a hazardous lesion because when eukaryotic replicative DNA polymerases (Pols) replicate DNA containing an 8-oxoG lesion they do so by inserting an A opposite the lesion (Einolf and Guengerich, 2001; Shibutani et al., 1991); which, results in G:C to T:A transversions.

DNA polymerases that belong to the Y-family promote the continuity of the replication fork by allowing replication through DNA lesions (Prakash et al., 2005). Humans have four Y-family polymerases – Polη, Polι, Polκ, and Rev1 – each with a unique DNA damage bypass and fidelity profile. Polι, for example, can proficiently incorporate nucleotides opposite N2-adducted guanines which project into the minor groove and opposite adducts such as 1, N6-ethanodeoxyadenosine which impair the ability of the purine to engage in Watson-Crick (W-C) base-pairing (Nair et al., 2005a, 2006; Nair et al., 2004; Washington et al., 2004; Wolfle et al., 2006). Rev1 is highly specialized for the incorporation of C opposite template G (Nelson et al., 1996; Haracska et al., 2002) and promotes efficient dCTP incorporation opposite bulky N2-dG adducts via a protein-template directed mechanism of DNA synthesis (Nair et al., 2005b, 2008). Polκ is specialized for the extension step of lesion bypass but can also preferentially insert an A opposite an 8-oxoG lesion (Lone et al., 2007; Vasquez-Del Carpio et al., 2009; Washington et al., 2002). In all, Y-family polymerases in eukaryotes display a large degree of functional divergence, rendering them highly specialized for specific roles in lesion bypass.

Polη is the only DNA polymerase demonstrated to act as a tumor suppressor in humans (Prakash et al., 2005). Polη is unique in its ability to replicate through an ultraviolet (UV)-induced cis-syn thymine-thymine (T-T) dimer by inserting two As opposite the two Ts of the dimer with the same efficiency and accuracy as opposite undamaged Ts (Johnson et al., 1999b). Because of the involvement of Polη in promoting error-free replication through cyclobutane pyrimidine dimers (CPDs), its inactivation in humans causes the variant form of xeroderma pigmentosum (Johnson et al., 1999a; Masutani et al., 1999), a genetic disorder characterized by a greatly enhanced predisposition to sun induced skin cancers. In addition to CPDs, Polη can also replicate through 8-oxoG lesions. In particular, yeast Polη replicates through 8-oxoG efficiently and accurately by inserting a C opposite the lesion and then proficiently extending from the 8-oxoG.C base pair (Carlson and Washington, 2005; Haracska et al., 2000). Genetic studies in yeast have provided support for the role of Polη in error-free bypass of 8-oxoG lesions (Haracska et al., 2000), as evidenced by a synergistic increase in the rate of spontaneous mutations in the absence of Polη in the ogg1Δ mutant yeast strain, defective in the glycosylase required for 8-oxoG removal.

To understand the basis of Polη’s ability to efficiently and accurately bypass 8-oxoG lesions, we determined the structure of yeast Polη in ternary complex with a template-primer presenting an 8-oxoG lesion in the active site and with dCTP as the incoming nucleotide (the ‘insertion” complex), and the structure of Polη extending from an 8-oxoG.C base pair (the “postinsertion” complex). Together, the two structures define for the first time the basis of Polη’s action on oxidatively damaged DNAs; revealing an active site that is well adapted to accommodate an 8-oxoG lesion at both the insertion and postinsertion steps of DNA synthesis in eukaryotic cells.

Structure determination

As shown previously, when we crystallized Polη with DNA it led to crystals that were of the same form as the previously reported apoenzyme (apo), and in which the DNA was often disordered or only partially bound (Silverstein et al., 2010). To cocrystallize Polη with an 8-oxoG lesion, we employed a strategy similar to the one used to cocrystallize Polη with UV-damaged and undamaged DNAs (Silverstein et al., 2010). That is, we made a K140A, S144W double mutant of Polη that breaks the “apo” crystals contacts but does not alter the function of Polη in vivo. Using the K140A, S144W protein, we were successful in obtaining cocrystals with 8-oxoG that diffracted to high resolution, belonged to a different space group, and possessed unit cell dimensions that were different from the apoenzyme. The insertion ternary complex cocrystals were obtained with a 16-nt/11-nt template/primer presenting the 8-oxoG lesion as the templating base and with dCTP as the incoming nucleotide. The postinsertion ternary complex cocrystals were obtained with a 16-nt/11-nt template/primer designed to “fix” an 8-oxoG.C base pair at the post-insertion template-primer junction, and with templating G opposite incoming dCTP. Both cocrystals diffract to 2.0Å resolution with synchrotron radiation, and the structures were determined by molecular replacement using the polymerase from the Polη ternary complex with undamaged DNA as a search model. Electron density maps showed clear densities for the bound DNAs, incoming dNTPs, and the 8-oxoG lesions (Fig. 1). For the insertion ternary complex, the final model includes residues −3 to 512 of the protein (residues −3 to 0 correspond to the linker region between Polη and the cleaved GST tag), nucleotides 1 to 11 of the primer strand, nucleotides 4 to 16 of the template strand, one dCTP molecule, two magnesium ions, three sulfate ions, and 705 water molecules. For the postinsertion complex, the final model includes residues −2 to 512 of the protein, nucleotides 1 to 11 of the primer strand, nucleotides 4 to 16 of the template strand, one dCTP molecule, two magnesium ions, three sulfate ions, and 601 water molecules.

Figure 1. Ternary structures of Polη 8-oxoG insertion and postinsertion complexes.

Figure 1

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(A) Ribbon diagrams depicting the Polη-DNA-dCTP ternary structures with 8-oxoG in the insertion position (left) and at the postinsertion position (right). The Polη palm, fingers, thumb domains and PAD are shown in cyan, yellow, orange, and green respectively. The DNA is in gray, and the putative Mg2+ ions are in dark blue. The 8-oxoG residues and incoming nucleotides are shown in red. (B) Simulated annealing Fo-Fc omit electron density maps contoured at 3.0 σ (resolution 2.0 Å) showing base-pairing between 8-oxoG and incoming dCTP in the insertion complex (left) and between 8-oxoG and the 3’ cytosine at the primer terminus in the postinsertion complex (right).

Overall Arrangement

The arrangement of Polη is very similar in the insertion and the postinsertion ternary complexes. In both structures, Polη embraces the template-primer with its palm (residues 1–30 and 130–286), fingers (residues 34–127), and thumb (residues 289–378) domains, as well as the polymerase associated domain (PAD; residues 395–509) unique to Y-family polymerases (Fig. 1) (Trincao et al., 2001). The palm carries the active site residues, Asp30, Asp155 and Glu156, which catalyze the nucleotidyl transfer reaction (Kondratick et al., 2001). The thumb and the PAD straddle the duplex portion of the template-primer, connected by a long linker that cradles one side of the DNA (Fig. 1). The thumb skims the minor groove surface, while the PAD is anchored in the major groove. The majority of DNA interactions are mediated by the PAD, wherein the main chain amides on the “outer” β-strands of the PAD β-sheet make a series of hydrogen bonds with the template and primer strands. In both structures, incoming dNTP binds with its triphosphate moiety interlaced between the fingers and palm domain, making identical hydrogen bonds with Tyr64 and Arg67 from the fingers domain and Lys279 from the palm domain, while the sugar packs against the aromatic ring of Phe35 (Fig. 2A). The catalytic residues Asp30, Asp155 and Glu156 are arrayed between the dNTP triphosphate moiety and the primer terminus, and two Mg2+ ions – analogous to metals “A” and “B” in replicative DNA polymerases (Doublie et al., 1998; Li et al., 1998; Steitz, 1999)– complete the Polη active site (Fig. 2A). Thus, Polη is well poised for dNTP insertion in both the 8-oxoG insertion and the postinsertion complexes. The putative 3’oxygen (at the primer terminus) is located ~3.3Å from the dNTP α-phosphate and aligned more or less linearly with respect to the Pα-O3’ bond (angle of about 155°) in each structure. The duplex portion of the template primer has a B-DNA-like conformation in both structures, with average helical twist and rise values of ~32.4° and 3.3Å, respectively.

Figure 2. Active site regions.

Figure 2

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(A) Close-up views of the active site regions within the Polη 8-oxoG insertion (left) and postinsertion (right) complexes. The palm and fingers domains and the PAD are colored cyan, yellow, and green, respectively. The incoming dCTPs and the 8-oxoG lesions at the T0 position (left) and the T1 position (right) are shown in red. The DNA is colored gray, and the putative Mg2+ ions are dark blue. Highlighted are the catalytic residues (D30, D155, and E156), residues interacting with the incoming dCTP (F35, Y64, R67, and K279), residues interacting with the templating base (Q55) and the 5’ nucleotide (R73 and M74), and residues interacting with the phosphate group of the T1 nucleotide (W56, N398, and N400). (B) Hydrogen bonding interactions between Polη residues Q55, W56, N398, and N400 and the T0 and T1 nucleotides in the 8-oxoG insertion (left) and postinsertion (right) complexes. A water molecule (magenta) that mediates interaction between N400 and the O8 atom of 8-oxoG in the postinsertion complex is also shown (right).

Insertion of C opposite 8-oxoG

The Polη active site is well-adapted to accommodate an 8-oxoG lesion in the anti conformation for W-C base pairing with incoming dCTP (Figs. 1B & 2A). The O8 of 8-oxoG (anti) does not sterically impinge on the active site residues and the closest residue from the PAD (Asn400) lies ~4.5Å away. Compared to the undamaged ternary complex, there is no major alteration in the polymerase structure (rmsd of 0.18Å over 510 Cαs) (Fig. 3A). The 8-oxoG lesion is stabilized in the active site cleft in part by a hydrogen bond between N3 of 8-oxoG and Nε2 of Gln55 from the fingers domain (Fig. 2B). There is an analogous hydrogen bond between O2 of templating T and Nε2 of Gln55 in the undamaged complex (Silverstein et al., 2010). Also, as in the case of undamaged complex, the nucleotide 5’ to templating 8-oxoG stacks over the templating base and makes a hydrogen bond with Arg73 and van der Waals contacts with Met74 (Figs. 2A & 3A). One difference is that whereas the 5’ nucleotide is in the syn conformation in the undamaged complex, it is in the anti conformation in the 8-oxoG complex. This is because Ile60 in the fingers domain adopts a different rotamer in the two complexes (Fig. 3A), making van der Waals interactions with the C5 methyl group of 5’T (syn) in the undamaged complex (Silverstein et al., 2010).

Figure 3. Comparison to the undamaged complex.

Figure 3

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(A) The active site region of Polη in the 8-oxoG insertion complex (blue) superimposed on the equivalent region in the undamaged ternary complex (gray). For clarity, only the DNA from the 8-oxoG insertion complex is shown, with the 8-oxoG lesion and incoming dCTP highlighted in red. (B) A portion of the template strand in the 8-oxoG insertion (blue) superimposed on the equivalent DNA segment in the undamaged ternary complex (gray). Highlighted are the distances from the O8 atom of the 8-oxoG lesion to the 5’ phosphate group in the damaged (4.3 Å; actual) and undamaged (3.9 Å; modeled) structures.

Surprisingly, the conformation of the template strand is relatively unaffected by the presence of 8-oxoG in the anti conformation (Fig. 3B). In all other DNA polymerase structures that have been determined with templating 8-oxoG in the anti conformation, the 5’ phosphate group of 8-oxoG is either kinked or rotated by 180° (Brieba et al., 2004; Freisinger et al., 2004; Krahn et al., 2003; Rechkoblit et al., 2006; Zang et al., 2006). This distortion in the DNA is thought to be necessary to circumvent a potential clash between the O8 of 8-oxoG and the sugar-phosphate backbone. In contrast, in Polη, the 8-oxoG backbone torsion angles α-ζ are close to the values observed in the undamaged complex, namely −61.0°(8-oxoG)/−73.5°(undamaged), 170.5°/178.5°, 49.4°/65.5°, 135.2°/136.5°, - 179.9.0°/−172.1°, −94.8°/−94.4°, respectively, which allow the 5’ nucleotide to continue its helical passage across the open Polη active site cleft (Fig. 3). However, these backbone torsion angles deviate significantly from those in “ideal” B-DNA (Saenger, 1984); for example, the α torsion angle (about the P-O5’ bond) in the Polη structures is more negative by ~30°, the β torsion angle (about the O5’-C5’ bond) is more positive by ~20°, and the γ torsion angle (about the C5’-C4’ bond) is more positive by ~20°. One consequence of these small deviations in the α–γ torsion angles (especially in α) is that the O2P atom is cast further away from the templating base in the Polη structures. Thus, whereas in ideal B-DNA the O8 of 8-oxoG would be ~3.4Å from the O2P atom, this distance increases to 3.9Å and 4.3Å in the Polη structures with undamaged and 8-oxoG templates, respectively (Fig. 3B).

Extension from 8-oxoG.C

In the postinsertion complex, an 8-oxoG (anti).C (anti) base pair is established at the T1P1 position (where T and P refer to template and primer strands, respectively, and the subscripts refer to the number of base pairs from the templating base position). The 8-oxoG lesion is stabilized in the anti conformation by a direct and a water mediated hydrogen bond between the O8 of 8-oxoG and Asn398 and Asn400, respectively, from the PAD (Fig. 2B). Other than these hydrogen bonds, there are no significant differences when the structure is compared to the undamaged complex or to the 8-oxoG insertion complex (Fig. 2). The 5’ phosphate of 8-oxoG is stabilized by hydrogen bonds with Nε2 of Trp56 stemming from the fingers domain and Nδ2 of Asn400 from the PAD (Fig. 2B), in much the same way as in the undamaged and the 8-oxoG insertion complexes. Also, templating G (at position T0) makes WC hydrogen bonds with incoming dCTP, as well as a hydrogen bond with Gln55 in the fingers domain (Fig. 2). In all, the polymerase superimposes with an rmsd of ~0.2Å (over 510 Cαs) when compared to the undamaged complex. As in the insertion complex, the 8-oxoG DNA backbone torsion angles deviate significantly from those in ideal B-DNA, with the distance between O8 of 8-oxoG and the 5’ O2P atom increasing to ~4.7 Å.

Discussion

8-oxoG adducts are formed frequently by the attack of oxygen free radicals on DNA. They are amongst the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions (Shibutani et al., 1991). We show here that the yeast Polη active site is well-adapted to accommodate 8-oxoG in the anti conformation, at both the insertion and the postinsertion steps of lesion bypass, which allows it to proficiently replicate through 8-oxoG by incorporating a C (Figs. 1–4).

Figure 4. Rotation of 8-oxoG from anti to syn conformation.

Figure 4

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(A) 8-oxoG (anti). dCTP (anti) hydrogen bonding in the insertion complex (left) compared to putative pairing between 8-oxoG (syn) and modeled dATP (anti) (right). 8-oxoG (syn) was generated by rotating the base around the glycosidic bond from χ = −90.2° to 88.9°. dATP was modeled onto dCTP via its sugar and triphosphate moiety. Note that oxidation of guanine to 8-oxoG results in the protonation of N7, changing this atom from a hydrogen bond acceptor to a hydrogen bond donor. For optimal 8-oxoG(syn).dATP(anti) base pairing dATP may need to adjust slightly “inward”. (B) Surface representations of the active site cleft region in the 8-oxoG (anti).C (anti) insertion complex (left) and when 8-oxoG is rotated to the syn conformation and paired with modeled dATP (anti) (right). The protein is colored in gray; incoming dCTP and dATP and the 8-oxoG lesions are colored in red; and the active site cleft residues (Q55, W56, R73, M74, N398, and N400) are colored in blue.

From pre-steady state kinetic studies, an 8-oxoG lesion does not impede yeast Polη at either the nucleotide binding or the incorporation step (Carlson and Washington, 2005). Polη binds dCTP opposite 8-oxoG with only ~2-fold lower affinity as opposite G, and the nucleotide is then incorporated at nearly the same rate as opposite G (Carlson and Washington, 2005). This is reflected in the nearly identical structures of Polη inserting a nucleotide opposite an 8-oxoG lesion or opposite an undamaged base. The geometry of the active site residues, the binding of the catalytic metals, and the position of the putative primer 3’OH (for the nucleotidyl transfer reaction) are almost indistinguishable in the 8-oxoG insertion and the undamaged ternary complexes.

Surprisingly, there is almost no change in the template strand in accommodating an 8-oxoG (anti) at the templating position (Fig. 5). In contrast, in all other DNA polymerase structures determined with templating 8-oxoG in the anti conformation, the 5’ phosphate group of 8-oxoG is either kinked or rotated by 180° (Brieba et al., 2004; Freisinger et al., 2004; Krahn et al., 2003; Rechkoblit et al., 2006; Zang et al., 2006). Polη differs from other polymerases in the “openness” of its active site cleft, allowing two unpaired template bases to be accommodated unhindered within the cleft. Because the unpaired 5’ base stacks directly above the templating base in the Polη active site cleft, any kinking or rotation of the sugar-phosphate backbone - of the type observed in phage T7 Pol (Brieba et al., 2004), mammalian Polβ (Krahn et al., 2003), and archaeal Y family Dpo4 (Fig. 5) (Rechkoblit et al., 2006; Zang et al., 2006), for example - would be unfavorable, resulting not only in the unstacking of the 5’ base but also in the loss of interactions with Arg73 and with Met74. Instead, when DNA binds Polη its backbone torsion angles appear to be “set” to accommodate the O8 of 8-oxoG (anti) without steric interference.

Figure 5. Comparison to Dpo4.

Figure 5

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DNA backbone changes in Polη (left) and Dpo4 (right) when inserting opposite 8-oxoG are shown in blue and orange, respectively, and undamaged bases are shown in gray. Highlighted are the distances between the O8 atom of the 8-oxoG residue and the location of the nearest phosphate groups in the undamaged (2.9 Å) and 8-oxoG insertion (4.9 Å) Dpo4 ternary structures (c.f. Figure 3).

From pre-steady state kinetic studies, Polη binds dATP opposite 8-oxoG with a similar affinity as dCTP, but the incorporation of A is ~100-fold slower than the incorporation of C (Carlson and Washington, 2005). From the structure, the open active site cleft of Polη permits easy interchange between 8-oxoG in anti and syn conformations for binding dCTP and dATP, respectively. That is, when we rotate 8-oxoG from anti (χ = −90.2°) to syn confirmation (χ = 88.9°) there is no steric overlap with any residues from the fingers or other domains (Fig. 4). However, a simple rotation about the glycosidic bond does not give rise to proper 8-oxoG(syn):dATP (anti) base pairing (Fig. 4); for example, the distance between modeled O6 of 8-oxoG(syn) and modeled N6 of dATP (anti) is 3.6Å. For optimal 8-oxoG(syn):dATP (anti) base pairing, dATP may move slightly “inward” and the accompanying repositioning of its α–phosphate may slow the rate at which dATP is incorporated opposite 8-oxoG(syn). Overall, the mechanism by which Polη selects C over A for incorporation opposite 8-oxoG appears to differ from that in other polymerases. For example, in phage T7 Pol, C is preferred over A because a lysine (Lys536) in the fingers domain sterically and/or electrostatically interferes with 8-oxoG when it is rotated from the anti to the syn conformation (Brieba et al., 2004; Brieba et al., 2005). Archaeal Dpo4, a homolog of Polκ, also prefers to incorporate C over A opposite 8-oxoG, but in this case, the anti↔syn equilibrium is tilted towards the anti conformation by favorable contacts that the polymerase makes with the O8 of 8-oxoG(anti) and the 5’phosphate that is flipped by 180° (Rechkoblit et al., 2006) In contrast to T7 Pol and Dpo4, Polη appears to select C over A not so much at the initial binding step but at the subsequent catalytic step.

Polη is efficient not only in inserting a C opposite an 8-oxoG lesion but also in extending from the resulting 8-oxoG.C base pair (Haracska et al., 2000). This dual proficiency is central to the ability of Polη to bypass 8-oxoG lesions in an error-free manner. From the structure, an 8oxoG(anti).C(anti) base pair at the postinsertion position does not affect the conformation of the polymerase or the bound DNA. The active site geometry is the same as that in the undamaged complex, allowing for efficient extension from a C base at the 3’ primer terminus. Archaeal Dpo4 is the only other polymerase, to our knowledge, that extends efficiently and accurately from an 8-oxoG.C base pair (Eoff et al., 2007; Rechkoblit et al., 2006; Zang et al., 2006). Intriguingly, structures of Dpo4 with 8-oxoG.A at the postinsertion position reveal a mixture of 8-oxoG(syn).A(anti) and 8-oxoG(anti).A(syn) base pairs (Rechkoblit et al., 2009), suggesting that the inefficiency of Dpo4 in extending from an 8-oxoG.A base pair is due to the resulting conformational flexibility of the primer terminus. Whether the inefficiency of Polη in extending an 8-oxoG.A base pair can be similarly explained by such conformational heterogeneity is unclear at present. However, the fact that 8-oxoG is stabilized in our structure in the anti conformation via interactions with residues (Asn398 and Asn400) from the PAD may allow for the unusual 8-oxoG(anti).A(syn) base pairing observed in the Dpo4 structures (Rechkoblit et al., 2009). In contrast to Polη and Dpo4, high fidelity polymerases such as phage T7 Pol or Bacillus Pol I fragment (BF) strongly prefer to extend from an 8-oxoG.A base pair (Brieba et al., 2004; Hsu et al., 2004). Unlike Polη and Dpo4, an 8-oxoG(anti).C(anti) base pair at the postinsertion position in the T7 Pol and BF complexes induces a local conformational changes in the template strand that propagates to the templating base and provides a rationale for the relative inefficiency of these polymerases in extending an 8-oxoG.C base pair (Brieba et al., 2004; Hsu et al., 2004).

We provide here for the first time a structural basis for the error-free replication of oxidatively damaged DNA by Polη. We show that the open active site cleft of Polη can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and the postinsertion steps of the lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Polη to prevent the mutagenic replication of 8-oxoG lesions.

Materials and Methods

Protein and DNA preparation

The catalytic core (residues 1–513) of S.cerevisiae Polη K140A S144W was expressed as an N-terminal GST-fusion protein in E. coli from plasmid pSL414 as previously described (Silverstein et al., 2010). Briefly, the GST-Polη1–513 K140A S144W fusion was expressed in E.coli and purified by affinity chromatography using a glutathione-Sepharose column. The GST tag was removed on the column by incubation with PreScission Protease, and the eluted Polη1–513 K140A S144W protein was further purified by ion exchange (Q Sepharose) before being concentrated for crystallization. The 11-nt DNA primer was synthesized with a 2’,3’-dideoxycytosine at its 3’-end (5’-GTCCTCCCCTCdd-3’). This primer was annealed to either a 16-nt template strand harboring a 7,8-dihydro-8-oxoguanine (8-oxoG) modification in the insertion position (5’-TAATGGAGGGGAGGAC-3’) or in the postinsertion position (5’-TAATGGAGGGGAGGAC-3’) (W.M. Keck, Yale). The primer and template strands were purified by ion exchange chromatography prior to annealing.

Cocrystallization

Both the insertion and the postinsertion ternary complexes were formed by incubating Polη1–513 K140A S144W with the respective primer-template in a molar ratio of 1:2 along with 10 mM dCTP and 10 mM MgCl2. Cocrystals of both the 8-oxoG insertion and the postinsertion ternary complexes were obtained from solutions containing 30% PEG 4000, 0.2 M Li2SO4, and 0.1 M TRIS (pH 8.5). (In contrast, we have been unable to obtain suitable cocrystals with incoming dATP.) For data collection, crystals of both complexes were cryoprotected by soaking them in mother liquor solutions containing increasing amounts of glycerol (0–15%) followed by flash-freezing in liquid nitrogen. Cocrystals of both complexes belong to space group C2221, with cell dimensions of a=88.0Å, b=227.6Å, and c=86.1Å for the insertion complex, and a=88.0Å, b=228.3Å, and c=85.9Å for the postinsertion complex (Table 1).

Table 1.

Data Collection and Refinement Statistics

Insertion complex Postinsertion complex
Data Collection
Wavelength (Å) 1.0809 0.9795
Space group C2221 C2221
Cell dimensions
     a, b, c (Å) 88.0, 227.6, 86.1 88.0, 228.3, 85.9
     α, β, γ(°) 90.0, 90.0, 90.0 90.0, 90.0, 90.0
Resolution (Å)a 50.0-2.0 (2.07-2.00) 50.0-2.0 (2.07-2.00)
Rsym (%)b 10.7 (61.3) 11.2 (57.6)
I/σ(I) 17.7 (3.0) 17.9 (3.7)
Completeness (%) 99.5 (94.1) 99.9 (98.4)
Redundancy 6.1 (6.0) 7.4 (7.5)
Refinement
Resolution (Å) 35.0-2.0 50.0-2.0
No. reflections 55,839 55,809
Rcrystc/Rfreed 15.7/19.5 15.8/19.2
No. atoms
     Protein 4,197 4,147
     DNA/dCTP/Mg2+/Sulfate 490/28/2/15 490/28/2/15
     Water 705 601
B-factors (Å2)
     Protein 22.4 24.3
     DNA/dCTP/Mg2+/Sulfate 32.8/16.0/27.7/34.9 33.8/16.7/31.8/51.7
     Water 35.0 35.2
R.m.s. deviations
     Bonds (Å) 0.014 0.014
     Angles (°) 1.59 1.58
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a

Numbers in parentheses represent values in the highest resolution shell.

b

Rsym = Σ|I - <I>|/ΣI, where I is the integrated intensity of a given reflection.

c

Rcryst = Σ‖Fobserved| - |Fcalculated‖/Σ|Fobserved|.

d

Rfree is calculated from 5% of the data excluded from refinement.

Structure determination and refinement

X-ray data on cryo-cooled 8-oxoG insertion and postinsertion cocrystals were measured at the National Synchrotron Light Source (beamline X29) and at the Advanced Photon Source (beamline 24-ID), respectively (Table 1). The data were indexed and integrated using DENZO, and scaled using SCALEPACK (Otwinowski and Minor, 1997). The program PHASER (McCoy et al., 2005) was used to obtain unique molecular replacement solutions using the apo-Polη structure (without the PAD) as a search model. PHASER was subsequently used to place the PAD as well. The solutions were refined, and the subsequent electron density maps showed clear densities for the respective primer-templates, incoming nucleotides, and metal ions. Both models were subjected to iterative rounds of restrained refinement with REFMAC (Winn et al., 2003) and building with Coot (Emsley and Cowtan, 2004). Protein residues 1–72, 73–150, 151–306, 307–388, and 389–512 and each individual DNA strand were treated as TLS groups, as identified using the TLSMD server (http://skuld.bmsc.washington.edu/~tlsmd/), during later rounds of refinement. TLS refinement reduced the Rfree of the 8-oxoG insertion complex to 19.5% with an Rcryst of 15.7%, and the Rfree of the 8-oxoG postinsertion complex to 19.2% with an Rcryst of 15.9%. The final refined model of the insertion complex includes residues −3 to 512 of the protein (residues −3 to 0 correspond to the linker region between Polη and the cleaved GST tag), nucleotides 1 to 11 of the primer strand, nucleotides 4 to 16 of the template strand, one dCTP molecule, two magnesium ions, three sulfate ions, and 705 water molecules. The final refined model of the postinsertion complex includes residues −2 to 512 of the protein, nucleotides 1 to 11 of the primer strand, nucleotides 4 to 16 of the template strand, one dCTP molecule, two magnesium ions, three sulfate ions, and 601 water molecules. Both structures have excellent stereochemistry, with 98.7% and 98.6% of the residues in the most favored regions of the Ramachandran plot for the insertion and postinsertion complexes, respectively, according to the program MolProbity (Davis et al., 2007). Simulated annealing omit maps were generated with CNS and all structural figures were generated with PyMOL (Delano Scientific).

Acknowledgments

We thank the staff at Brookhaven National Laboratory (X29) and the Advanced Photon Source (24ID) for facilitating X-ray data collection. We thank D.T. Nair, S. Lone, R. Vasquez-Del Carpio, and M. Swan for help and discussions. This study was supported by NIH grants ES017767 and CA107650.

Footnotes

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Accession Numbers

The coordinates and structure factors have been deposited in the protein data bank with accession codes 3OHA and 3OHB for the insertion and postinsertion complexes, respectively.

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