Kimberly (Wei-Wei) Oo

Harvard College ‘09,


The cell has developed a number of defenses against DNA damage; glycosylases, for example, remove damaged bases. Human 8-oxoguanine glycosylase 1 (hOGG1) is responsible for the excision of the damaged base oxoguanine (oxoG). In this study, we present the first reported structure of hOGG1 containing guanine in a fully wild type active site. In addition, we also present the structure of a novel catalytic intermediate during the excision of oxoG.


Maintaining the integrity of the genome is crucial for the survival of an organism. DNA is vulnerable to damage from a variety of sources, both extrinsic and intrinsic to the cell (Lindahl 1993; Friedberg 2003). Unless the cell can repair this damage, it will cause mutations in the genome. Thus, DNA repair mechanisms in the cell are extraordinarily important (Lindahl 1993). One common kind of DNA damage is the oxidation of guanine to 8-oxoguanine (oxoG) by reactive oxygen species (Neeley 2006; Henle 1997), which are commonly produced by ionizing radiation, exposure to transition metals, or even as byproducts of aerobic respiration (Henle 1997; Bjelland 2003). Although the Watson-Crick face of oxoG is identical to that of guanine and can still base pair with cytosine, it can also rotate around its glycosidic bond and base pair with adenine through a Hoogsteen interaction (Oda 1991) (Figure 1). During replication, DNA polymerase preferentially adds adenine across from oxoG, which after two rounds of replication, causes a G:C to T:A transversion, thereby introducing a mutation into the genome (Hsu 2004; Shibutani 2001).

Figure 1. The cause of the mutagenic potential of oxoG. Reproduced from Crenshaw 2009.

Human 8-oxoguanine glycosylase 1 (hOGG1) locates and excises oxoG from the genome as part of the base excision repair pathway (Crenshaw 2009; David 1998). hOGG1 is a bifunctional enzyme, acting as a glycosylase, which breaks the N-glycosidic bond to the base, and a β-lyase, which nicks the DNA on the 3′ side of the sugar (Boiteux 2001). The enzyme diffuses along the DNA until it locates oxoG. It induces a bend in the DNA and extrudes oxoG out of the DNA helix and into its active site (Bruner 2000). The structural intermediates involved in flipping out the base from the DNA helix make up the base extrusion pathway. Then, hOGG1 excises oxoG, creating an abasic site, and nicks the DNA (Dodson 1994). Other members of the base excision repair pathway then remove the sugar and insert a new guanine nucleotide, repairing the DNA (Vidal 2001).

One goal of the Verdine lab is to answer the “search problem,” namely, how hOGG1 distinguishes oxoG from other bases. Although hOGG1 can distinguish between damaged and normal bases while they are within its active site through specific contacts, it is unclear whether it is necessary to individually flip out each base into the active site to find rare oxoG substrates hidden in the genome (Bruner 2000). Such a process seems unlikely since hOGG1 slides along DNA at a velocity approaching the upper limit for one-dimensional diffusion (Blainey 2006). However, when oxoG is intrahelical, there are no obvious structural distortions to the DNA duplex that could be used to distinguish between oxoG and guanine (Bowman 2008; Lipscomb 1995; Oda 1991).

To determine if hOGG1 can distinguish between guanine and oxoG while the bases are intrahelical, it would be ideal to capture “encounter complexes” of hOGG1 on an intrahelical oxoG and guanine. With these structures, one could compare the interactions between the protein and DNA to determine if the interactions differed between oxoG and guanine. Thus, our initial goal was to solve the structure of hOGG1 with an intrahelical guanine.

Another goal of our lab is to elucidate the catalytic mechanism hOGG1 uses to cleave out oxoG. In general, a bifunctional glycosylase uses a nucleophilic residue to displace the undesired base from the sugar, opening the ring and forming a Schiff base. β-elimination of the 3′ phosphate through deprotonation of the C2′ hydrogen cleaves the DNA strand and forms an α, β unsaturated Schiff base (David 1998), which is then hydrolyzed to release the glycosylase from the DNA, completing the mechanism. The structures of several intermediates have been solved to bring insight into the specific mechanism hOGG1 uses (Fromme 2003; Radom 2006; Chung 2004).

Here, we present two crystal structures of hOGG1-DNA complexes. The first is a structure of hOGG1 containing guanine within its active site, the “G complex.” This structure was solved while we attempted to solve a structure of hOGG1 with an intrahelical guanine. The G complex was completely unexpected because previous studies indicated that guanine would be excluded from the active site (Banerjee 2006). In spite of being in the active site of a catalytically active hOGG1, the guanine was not excised out of the DNA. Thus, this study presents the first structure of hOGG1 with an undamaged base in the wild-type active site. The second structure presented is a structure of hOGG1 bound to a trans-α,β-unsaturated aldehyde, a previously unreported intermediate along the catalytic pathway.


Because hOGG1 has no specific preference for an undamaged base, to capture a structure with guanine it is necessary to restrict hOGG1 to that guanine so that the protein-DNA complex would be homogeneous enough for crystallographic studies. We formed a disulfide bond between a mutated cystine on hOGG1 and a modified thiol-containing base in the DNA, creating a disulfide crosslink that covalently tethers the two together, restricting hOGG1 to the desired base (Banerjee 2005) (Figure 2). This disulfide crosslink was necessary to determine the structure of many of the intermediates captured thus far (Banerjee 2005; Banerjee 2005; Radom 2007). Our lab has also employed this strategy to determine the structure of other systems suffering from similar problems (Huang 2000; Huang 1998).

Figure 2. Functionalization of adenine and crosslinking with hOGG1 at the S292C site

A number of intermediates along the base extrusion and catalytic pathways have already been captured. In the base extrusion pathway, crystal structures have been solved containing: guanine extruded out of the helical stack, but folded back into the major groove of the DNA (intermediate 1) (Banerjee 2006); oxoG (int. 2) (Radom 2007) and guanine (int. 3) (Banerjee 2005) in an “exo site,” with the base extrahelical, but on a part of hOGG1 adjacent to the active site; oxoG entering the active site (int. 4) (Radom 2007); and oxoG fully in the active site (int. 5-8) (Bruner 2000; Banerjee 2005; Radom 2007; Norman 2003). An overview of the previously captured intermediates in the base extrusion pathway is summarized in Figure 3.

Figure 3. An overview of the previously captured intermediates in the base extrusion pathway. hOGG1 is shown in purple, DNA in blue, and the interrogated base in red. The intermediates captured thus are categorized by: the location of the interrogated base; the crosslinking site, if there is one; and other mutations/information. PDB accession codes: 1.2I5W29, 2. 2NOF31, 3. 1YQK30, 4. 2NOZ31, 5. 1EBM15, 6. 1YQR30, 7.2NOL31, 8. 1N3C32.

In the earliest intermediate so far crystallized in the pathway, (int. 1), guanine was extruded out of the helical stack into the major groove (Banerjee 2006). The guanine was placed adjacent to oxoG, but a disulfide crosslink formed between N149C and the cytosine opposite from the guanine was short enough to restrict hOGG1 to interrogate the guanine. The crosslink forced the guanine out of the DNA helix because the crosslink physically occupied the same place the intrahelical guanine would have occupied. The extruded guanine formed a noncannonical base pair with the neighboring oxoG, stabilizing guanine in the major groove. Likewise, in the structure containing guanine in the exo site, (int. 3), the same disulfide crosslinking site was used, which biased the guanine to be extrahelical (Banerjee 2005).

These two results seemed to imply that when forced out of the helix, guanine should have no strong preference for any particular site extrahelical to the DNA, as both the exo site and the noncannonical base pair do not seem offer a particularly large amount of stabilization (Banerjee 2005; Banerjee 2006). Thus, it is likely that it is more favorable for guanine to be within the DNA helix, where there are specific interactions stabilizing it. As a result, moving the crosslink to a different site that would not sterically displace guanine from the DNA helix might allow the capture of a structure containing an intrahelical guanine (Radom 2006).

In this study, the crosslinking site was between S292C and an adenine four bases down from the guanine. This site had previously been employed to solve the late-stage intermediate structure (int. 4) with oxoG partially in the active site of hOGG1 (Radom 2007). In addition, the structure of a catalytically inactive mutant of hOGG1 crosslinked to oxoG containing DNA at the S292C site (int. 7) is almost completely identical to the structure of the mutant without the crosslinking site (int. 5) and the structure of the mutant with the N149C crosslinking site (Bruner 2000; Radom 2007) . Thus, the S292C crosslinking site seemed like it might not force the guanine to be extrahelical. As a result, we hoped that crystallization of hOGG1 crosslinked through S292C to guanine-containing DNA would allow the structural determination of a complex containing an intrahelical guanine. In reality, the structure captured was that of hOGG1 with guanine in its active site. An overview of the disulfide crosslinking strategy is presented in Figure 4.

Figure 4. An overview of the crosslinking strategy. A) Crystal structures of: (int. 1). K249Q hOGG1 (green) bound DNA (blue) with oxoG (red); (int. 9). S292C hOGG1 (purple) crosslinked to DNA with guanine (green); (int. 3). N149C hOGG1 (yellow) crosslinked to DNA with guanine. B) and C) A comparison of the S292C and N149C crosslink sites.

Several intermediates along the catalytic pathway have also been captured. An overview of the theorized catalytic mechanism summarizing the previously captured intermediates is presented in Figure 5. Two intermediates in the catalytic mechanism for hOGG1 have been captured using the technique of borohydride trapping (Fromme 2003; Radom 2006). Intermediates containing iminium ions can be reduced by sodium borohydride to the amine, halting catalysis at a particular step and allowing for the characterization of a normally fleeting intermediate (Zharkov 2002; Gilboa 2002). In hOGG1, the ε-amino group of Lys 249 is a nucleophile and displaces the oxoG base to create the initial Schiff base (II). This intermediate has been captured by treatment with sodium borohydride and the structure determined by X-ray crystallography (III) (Fromme 2003). In addition, the intermediate after β-elimination – the α, β unsaturated Schiff base (IV) – was captured by treatment with sodium cyanoborohydride (V) (Radom 2006).

Figure 5. An overview of the catalytic pathway of hOGG1. Red indicates that the structures have been crystallized. The geometry of double bond in IV and V can be either cis or trans. PDB accession codes: I. Same as (int. 5)-(int. 8), III. IHUO, V. No submitted, and VII. 1M3H.

Although borohydride trapping has been useful in determining the structures of intermediates, one limitation of borohydride and other chemical trapping methods is that they can only capture intermediates with certain functional groups. A more general technique is needed to discover all the intermediates in the catalytic cycle. To address this, Drs. Chris Radom and Seongmin Lee in the Verdine lab developed a “time resolved” crystallography technique (Radom 2006). Instead of chemically trapping an intermediate, the intermediate would be freeze-trapped by cryoprotection of crystals in liquid nitrogen. In brief, DNA containing a bulky photocleavable adduct attached to oxoG was crosslinked to hOGG1 by a disulfide tether between N149C and the cytosine opposite from oxoG and crystallized. The structure of a complex containing this bulky adduct has been solved, indicating that the bulky adduct prevents oxoG from entering the active site and instead places it at the exo site. Flashing the crystal with ultraviolet light cleaves the adduct off and allows oxoG to enter the active site of hOGG1. Therefore, all the hOGG1 synchronously begins excising the oxoG. After a certain length of time, the crystal is frozen with liquid nitrogen, and the low temperature (77K) prevents further catalysis from occurring. As crystals are usually cryoprotected to protect against radiation damage and increase the diffraction quality and resolution, this method of freeze-trapping the crystals is very convenient (Rodgers 1994; Henderson 1990). Ideally, this allows one to capture the various intermediates along the catalytic pathway by varying the time between photocleaving the adduct and freezing the crystal (Radom 2006).

This technique has been used to capture a late stage intermediate in the base extrusion pathway (Lee 2008). A crystal was cryoprotected immediately after photocleavage of the adduct, and the structure of that complex was solved. The oxoG was almost fully inserted into the active site of hOGG1, but the active site had not yet made any of the contacts with oxoG that have been observed in other structures containing oxoG in the active site. Thus, this structure validates this technique as a way to capture otherwise fleeting intermediates.

This study used the time resolved crystallography technique to solve the crystal structure of a catalytic intermediate obtained by freezing the crystal 30 minutes after photocleavage. It is hereafter referred to as the “30 minute structure.”

Results and Discussion

Crystallization of the G complex

The crystallization of the G complex was surprisingly difficult. The previous structures of base extrusion intermediates had all been crystallized in similar conditions, approximately 150 mM calcium chloride, 17% polyethylene glycol 8000, and 100 mM sodium cacodylate pH 6.0 within the 24 well hanging drop vapor diffusion method (Bruner 2000; Banerjee 2005; Banerjee 2006). However, when these conditions were used, the crystals were either too thin to be useful in X-ray crystallography or severely branched. In addition, they were fragile and broke apart while being transferred to cryoprotectant.

The screens were broadened to look at the effects of different salts (magnesium acetate and magnesium chloride), temperatures (4 and 20 °C), drop volumes, and protein complex to well solution ratios, without much change in crystal behavior. In addition, other crystallization techniques were tried, such as sitting drop vapor diffusion, the use of oils to slow vapor diffusion, macroseeding, streakseeding, and addition of additives, without much improvement.

To find a different crystal form that might be more amenable to crystallographic studies, ten 96-well Nextal screening suites (Qiagen) were used to screen the hOGG1 complex over a variety of different conditions. However, the crystals that resulted from this large-scale screen were almost all either needles or dendrites and were also difficult to reproduce in the 24-well format. As a result, this effort did not yield any useful results.

Eventually, a crystal yielding diffraction data of sufficient quality was produced. The complex was crystallized with the addition of additives in a hanging drop vapor diffusion format with the 24 well OptiSalts Screen (Hamptom). The resolution of the structure from this crystal was 3.05 Å. Unfortunately, reproduction of this condition did not produce higher resolution data, so the 3.05 Å structure was used.

Structure of the G complex

Charisse Crenshaw collected X-ray diffraction data for the G complex and scaled it to 3.05 Å (Figure 6). She solved the structure using the phases calculated from a previously solved hOGG1 structure, the K249Q, Q315A mutant containing oxoG in the active site (PDB accession code: 2NOH) (Radom 2007). Surprisingly, this structure was almost identical to structures containing oxoG in the active site, except that guanine was in the place of oxoG (heavy atom RMSD=0.401 Å with (int. 5), the structure of catalytically inactive hOGG1 (K249Q) bound to oxoG containing DNA) (Bruner 2000).

Figure 6. Data collection and refinement statistics of the G complex. Reproduced from Crenshaw 2009.

Conformational Changes

When hOGG1 binds oxoG in its active site, it undergoes conformational changes that allow its residues to make contact with oxoG (Crenshaw 2009; Radom 2007). In a structure without oxoG in the active site, such as in the structures along the base extrusion pathway, the α-O helix is held away from the active site in an “open” conformation (Banerjee 2005; Radom 2007). In this conformation, His 270 on the α-O helix forms a salt bridge with Asp 322 and an aryl-π interaction with Phe 319 (Figure 7a). However, in a structure containing oxoG in the active site, such as the structures captured with catalytically inactive mutants or the structures of the catalytic intermediates, the α-O helix moves towards the active site into a “closed” conformation, and His 270 instead forms a salt bridge with a phosphate of the DNA backbone, freeing Phe 319 to form a π-stacking interaction with oxoG. Furthermore, Gln 315 hydrogen bonds with the Watson-Crick face of oxoG (Figure 7b) (Bruner 2000; Radom 2007).

Figure 7. A comparison of the open and closed active sites. The G complex is purple. A) open active site (int. 3) is orange. B) closed active site (int. 5) is green.

In the structure of the active site G complex, the aforementioned residues occupy the same positions as the residues in the “closed” conformation, with the exception of His 270, which is rotated 90°. However, this rotation does not affect its ability to form a salt bridge with the DNA phosphate, and it functions in the same manner as the histidine in the “closed” conformation.

Discrimination between oxoG and guanine

The previous interactions are characteristic of the “closed” conformation and stabilize both oxoG and guanine. However, based on quantum mechanical calculations using (int. 3) and (int. 5), there are two sources of discrimination between oxoG and guanine in the active site of hOGG1 that might destabilize guanine binding in the active site (Banerjee 2005). First, there is a hydrogen bond between the Gly 42 carbonyl and the oxoG N7 that is lost with guanine. This is calculated to stabilize oxoG by about 3.5 kcal/mol. Second, the salt bridge between Lys 249 and Cys 253 causes a dipole that interacts favorably with the dipole of oxoG. However, since guanine has the opposite dipole, this would be a repulsive interaction, favoring oxoG by about 3.3 kcal/mol.

In the active site G complex, the positions of Gly 42, Lys 249 and Cys 253 stay roughly the same, along with the rest of the residues in the active site, when compared to (int. 5) (Figure 8). The loop containing Gly 42 was previously found to be rigid, which may explain why it does not move to relieve the posited repulsion. However, the distance between the Cα carbonyl of Gly 42 and N7 increases slightly (2.7 Å in (int. 5) to 3.2 Å in the active site G complex) by the base shifting down, which may relieve some repulsion. In addition, it is possible that the N7 of guanine is protonated, which would recreate a favorable interaction. This is unlikely, however, because N7 is not basic and is unlikely to be perturbed enough to be protonated. The other structures containing oxoG in the active site have distances similar to that of (int. 5): 2.9 Å (int. 6) (Banerjee 2005), 2.8 Å (int. 7) (Radom 2007) and 2.7 Å (int. 8 ) (Norman 2003), indicating that the increased distance to 3.2 Å may be significant. The last structure (int. 7) indicates that the increase is not directly due to the crosslinker, though it does not rule out a more subtle effect.

Figure 8. The effect of the S292C crosslink. A) and B) (int. 1) pink, (int. 3) yellow, (int. 8 ) green, (int. 5) blue, and (int. 6) purple. The length of the crosslink correlates with the position of the base along the base extrusion pathway. C) and D) The lowest energy conformations of the crosslink used at the S292C site. Figure reproduced from Crenshaw 2009.

The salt bridge also appears to be intact in the G complex. The distance between Lys 249 and Cys 253 increases slightly from 2.6 Å in (int. 5) (Bruner 2000) to 2.8 Å in the G complex, which is still consistent with a salt bridge (Petsko 2003). Thus, it is unlikely that these repulsive interactions were completely removed, though the repulsion may be lessened slightly. Therefore, it is unclear as to why guanine would be inserted in the active site, as is seen in this structure.

Crosslinking bias

The authors presenting (int. 2) and (int. 4), which contain oxoG in the exo site or partially in the active site, respectively, posed a similar question (Radom 2007). When the crosslinking site was moved from N149C to S292C, the oxoG moved further along the extrusion pathway. It was proposed that the crystal packing of the particular crystal form of (int. 4) bends the DNA to favor an extrahelical base (Radom 2007; Radom 2006). However, this leaves unresolved why in (int. 4), oxoG moved further along the base extrusion pathway instead of remaining in the exo site, as presumably the crystal forms and packing interactions were the same (since the crystallization conditions were similar and the space groups identical).

Here, we propose that DNA bending is indeed the key difference but that the DNA is bent because of the crosslinker, not because of crystal packing. The distance between S292 and the adenine, which is the distance a crosslinker would need span if it were placed at the S292C site varies depending on the stage of the base extrusion pathway (Figure 9). The structures of the early intermediates (1 and 3) require a larger span (10.1 Å and 9.6 Å), while the structures of later intermediates (5, 6, and 8) require a smaller span (8.1 Å and 8.3 Å). However, in the nine lowest energy conformations of the four-carbon crosslinker used at the S292C site, the largest span is 8.46 Å, which is too short to span the distances of the early intermediates (Figure 8). Thus, it is likely that the early intermediates, such as the exo site or intrahelical intermediates, are disfavored by the S292C four-carbon crosslinker, forcing the base into the active site.

Furthermore, in the previously solved structures containing the S292C crosslinker, (int. 4) and (int. 7), the distances spanned are 8.32 Å and 6.98 Å respectively. In the latter case, it is well under the maximum distance, explaining why there was no observable perturbation of that structure when compared to (int. 5), the structure without a crosslink.

Based on this evidence, it is probably not possible to capture an intrahelical base with a four-carbon crosslink, and a longer one is necessary. This also raises the possibility that different crosslink lengths can be used to capture different stages along the base extrusion pathway, though care must be taken to ensure that the intermediates captured are biologically relevant.

Synthesis of a C8 crosslink

An 8 carbon (C8) crosslink was synthesized to test this possibility. However, solubility problems prevented it from being able to functionalize the DNA. C8 was activated with Aldrathiol to reduce the size and make it less nonpolar. However, this was difficult to purify and was not ultimately used, as a shorter crosslink, such as a 5 or 6 carbon crosslink, would probably be a simpler and equally useful tool. (See Supplementary Information for more detail.)

Structure of the 30 minute structure

The X-ray diffraction data for this structure was collected, scaled to 2.44 Å, and solved (Figure 9). Overall, this structure resembles previously solved catalytic intermediates. Several characteristics of this structure should be noted. First, oxoG is not present in the active site. Second, C3′ and the 3′ phosphate are not covalently bound. Third, C1′ and Lys 249 are not covalently bound. Fourth, the C2′-C3’double bond is trans. Fifth, the structure is in the “open” conformation associated with base extrusion intermediates, instead of the “closed” conformation associated with catalysis. The first four characteristics point to this structure being a late stage intermediate in catalysis, after the hydrolysis of the Schiff base, but before the isomerization to the end-product structure (Figure 10). However, the fifth characteristic seems to contradict this conclusion.

Figure 9. Data collection and refinement statistics of the 30 minute structure. Figure reproduced from Crenshaw 2009.

A late stage intermediate

Most of the characteristics of this structure favor it as a late stage intermediate. During refinement, when oxoG was modeled into the active site, a strong negative density resulted in the σA-weighted fo-fc electron density maps, indicating that there is not enough electron density to indicate that oxoG is in the active site (data not shown). Instead, three water molecules were placed in the active site. The existence of water molecules within the active site in the absence of oxoG is consistent with other structures, including catalytic intermediates (III) (Radom 2006), (V) (Radom 2006), and (VII) (Chung 2004) and base extrusion intermediates (int. 2) (Radom 2007) and (int. 3) (Banerjee 2005).

In addition, there is no electron density between the C3′ and the 3′ phosphate. Furthermore, the distance between C3′ and the oxygen of the 3′ phosphate is 5.7 Å, much larger than a normal C-O bond. Thus, in this structure, C3′ and the oxygen of the 3′ phosphate are no longer covalently attached and β-elimination of the phosphate has already taken place (Figure 10).

Similarly, there is no electron density between C1′ and Lys 249, and the distance between the terminal amine and C1′ is 4.4 Å, in contrast with the normal distance of a C=N bond. Thus, lysine 249 is no longer involved in a Schiff base with C1′ and has been hydrolyzed off the sugar (Figure 10).

Figure 10. The active site of the 30 minute intermediate. In purple is the α,β-unsaturated aldehyde that formed from the sugar of oxoG.

Taken together, these three details of the structure indicate that this intermediate is after (IV) in the catalytic pathway. Thus, this structure is the latest intermediate currently captured along the catalytic pathway. Furthermore, this structure provides evidence that hOGG1 is able to catalyze the excision of oxoG even when it is constrained by crystal packing forces.

The geometry of the C2′-C3′ bond

In (III), the structure of the reduced initial Schiff base, the oxoG is already excised but remains bound within the active site of hOGG1, and its N9 is positioned within 4 Å of C2′ – a plausible distance for it to deprotonate C2’ (Fromme 2003). Furthermore, addition of oxoG analogues 8-bromoguanine (8-bromoG) and 8-aminoguanine (8-aminoG) can accelerate strand scission of an abasic site. X-ray crystal structures of the initial borohydride trapped Schiff base with the free oxoG purified out and 8-bromoG or 8-aminoG soaked into the hOGG1-DNA complex show that 8-bromoG and 8-aminoG occupy the same position that oxoG occupies in (III), indicating that oxoG may behave similarly to 8-bromoG and 8-aminoG (Fromme 2003). Taken together, this indicates that oxoG likely deprotonates C2′ and causes the elimination of the 3′ phosphate.

Molecular modeling calculations have indicated that the deprotonation of the C2′ proR hydrogen by oxoG is favored (80% proR to 20% proS) (Fromme 2003). Nevertheless, it is unclear if elimination of the hydrogen would lead to a cis- or trans-α,β-unsaturated Schiff base. Because the phosphate is antiperiplanar to the proR hydrogen, it is possible that the elimination is concerted and more E2-like, and thus the elimination of the proR hydrogen would lead to a trans-α,-β-unsaturated Schiff base. However, it is also possible that the elimination instead forms the enamine first and then eliminates the phosphate (thus is more E1cb-like), which may lead to either the trans or cis product. In addition, molecular modeling found that the trans product is more stable than the cis product, which is consistent with elimination reactions in general, but although this supports the trans product, it is possible that this reaction is under kinetic control (Fromme 2003). Furthermore, although the end-product structure (VII) contains the sugar is in the ring-closed form, and thus the double bond between C2′ and C3′ must be cis eventually, this does not preclude the formation of a trans double bond in the initial elimination, since this bond can later isomerize to the cis form (Chung 2004). Unfortunately, the crystal structure containing the α,β-unsaturated Schiff base caught by borohydride trapping (V) was similarly inconclusive. Models containing both the cis, trans, or saturated (from potential overreduction by sodium cyanoborohydride) bonds fit into the electron density of that structure, with little difference in the Rfree, so it was not possible to distinguish between the possibilities (Radom 2006). Complicating the matter, it is also possible that hOGG1 may produce a mixture of both the trans or cis products, which may be the reason for the ambiguity of (V).

The structure presented in this study resolves this issue. In this structure, the bond between C2′ and C3′ was determined to be trans. The sugar of oxoG could be in one of three conformations: cis, trans, or ring closed. To determine which of the sugar forms was likely, we modeled in the three different forms, and after performing one round of energy minimization and individual B factor refinement in CNS (Brunger 1998; Brunger 2007), found that the trans-α,β-unsaturated aldehyde best fit the electron density (Figure 11). In addition, the Rfree values after the round of refinement were 0.2602 for the trans, 0.2613 for the cis, and 0.2630 for the ring closed, indicating that of these three, the trans best described the structure. In addition, even though the cis fit the second best, it is likely that the cis intermediate may be too short lived to capture, because it can very favorably turn into the ring closed form since it is already in the conformation to do so. Thus, the comparison is between the trans and ring closed forms, and the trans intermediate fits the best.

Figure 11. Electron density maps of the 30 minute structure. A) The trans product is purple. B) The cis product is green. C) The ring closed product is yellow. Maps are σA-weighted 2Fo-Fc map contoured at 0.6 σ.

Thus, the bond between C2′ and C3′ appears to be trans, implying that the β-elimination of the 3′ phosphate occurs to give the trans product. Furthermore, this structure also indicates that isomerization of the trans to the cis product occurs on the aldehyde and not the Schiff base, since in this structure the Schiff base has already been hydrolyzed.

It is likely that this isomerization occurs through a conjugate addition of a nucleophile, and then the subsequent rotation and conjugate elimination of that nucleophile to form the cis-α,β-unsaturated aldehyde (Figure 12B). Although there has been no direct evidence of this isomerization, the existence of a religated product (VIII) formed from the addition of 8-aminoG to a crystal containing the end product structure (VII) implies that the phosphate is able to undergo a conjugate addition, albeit assisted by 8-aminoG (Figure 12A) (Chung 2004). Thus, it is possible that phosphate or a different nucleophile such as water, may be able to undergo conjugate addition if driven by an ultimate thermodynamic preference for the ring-closed structure.

Figure 12. A possible isomerization mechanism. A) This is a proposed mechanism for the formation of VIII (PDB accession code 1M3Q39) from VII. B) This is the proposed mechanism for the formation of VII from VI.

The “open” conformation

Surprisingly, this structure is in the “open” conformation. The α-O helix is held away from the active site and His 270 is interacting with Asp 322 and has an edge-face interaction with the π-system of Phe 319, which moves it away from the position it occupies in the “closed” conformation. In addition, Gln 315 is also away from its position in the “closed” conformation. In contrast, the previous two catalytic intermediates were in the “closed” conformation (Figure 13).

Figure 13. The 30 minute structure is in the open conformation. The 30 minute structure is in blue, with the sugar from oxoG in purple. III, a closed structure, is in green, with its oxoG and sugar in orange. Here π interactions are shown with dashes.

At this time, it is unclear whether the “open” conformation of the 30 minute structure is the natural conformation or if it has been artificially introduced by the system. Biochemical experiments done by Charisse Crenshaw have indicated that the disulfide crosslink, which is one of the differences between this structure and the previous catalytic intermediates, does not seem to have a significant affect on the conformation of the DNA, in contrast to the role it played in the G complex. However, this does not preclude bias introduced from other aspects of the freeze trapping system or from another unidentified bias from the crosslinker.

The previous two structures obtained using this freeze trapping technique were in the “open” conformation; however, it is unclear if this is because the structures were within the base extrusion pathway or if this system biases hOGG1 to the “open” conformation. In addition, it is possible that the “closed” conformation is only necessary for the initial part of catalysis and the conformation may move to the “open” form later during catalysis. Because this structure does not contain oxoG in the active site, the π-stacking interaction between Phe 319 and oxoG, which stabilizes the “closed” structure, is lost. Thus, it is possible that by this stage in the reaction, hOGG1 has moved back into the “open” form.

Moreover, although the end-product structure, which would be after this intermediate, does contain hOGG1 in the “closed” form (Chung 2004), this may be due to the mutation used to crystallize that structure, D268E, which had previously been known to affect the nearby residues 269-271 (Norman 2003). Because the interaction between His 271 and the DNA is a key part of the “open” conformation, the mutation may be biasing the structure of the molecule to the “closed” form. Again, however, it is not clear whether the “open” conformation is an accurate description of this intermediate or if it is an artifact brought on by the system used.


In the active site G complex, guanine is not cleaved out, even though the protein is catalytically active, and the active site of the guanine is in the “closed” conformation and presumably ready for catalysis. This is surprising because excision of oxoG by hOGG1 occurs within the 30 minute structure. The G complex and the 30 minute structure are in the same crystal forms and are thus presumably affected by the same crystal packing forces. Furthermore, the S292C crosslink – at least by itself – does not prevent catalysis, as hOGG1 with the S292C crosslink has been shown to excise oxoG in biochemical studies (Crenshaw 2009). However, guanine does not seem to have been excised, as the N glycosidic bond and the 3′ and 5′ phosphodiester bonds are all intact. In addition, biochemical experiments have shown that hOGG1 with the S292C crosslink does not excise guanine.

Thus, the G complex raises the possibility that there is a “catalytic checkpoint,” or a way that hOGG1 can prevent excision of guanine even in the likely rare case that guanine is placed in the active site. Such a catalytic checkpoint is not a ubiquitous feature of glycosylases: Uracil DNA glycosylase, (which sterically excludes thymine from entering the active site) when modified to allow thymine to enter the active site, will excise thymine (Bennet 2006). However, human thymine DNA glycosylase does use a catalytic checkpoint. It allows both thymine from T:G mismatches and cytosine from C:G matches into the active site but only excises thymine because thymine is a better leaving group than cytosine (Otwinowski 1997). OxoG is intrinsically a better leaving group than guanine, and it is possible that hOGG1 discriminates between oxoG and guanine at the level of catalysis, and not only during the search process.

The 30 minute structure is a previously uncharacterized late stage intermediate in the catalytic mechanism of oxoG excision. In addition, this intermediate confirms that elimination of the phosphate creates the trans α, β unsaturated Schiff base. It also lends support to the validity of the freeze trapping technique. However, in order to confirm whether the freeze trapping technique is valid, the structures of the other crystals for which data has already been collected should be solved. It is important to confirm that the stage in catalysis the intermediates are captured at correlates with the interval between photocleavage of the adduct and cryoprotection; if not, the progression of catalysis is too stochastic, and thus the crystals are likely to be too heterogeneous to be characterized accurately. Additionally, it would be useful to ensure that early intermediates captured using this methodology are in the “closed” form, like the other early intermediates; otherwise, it would indicate that the freeze trapping system artificially warps the structures.

Materials and Methods

For full methods, see the supplementary information online. In brief, hOGG1 was expressed in E. coli and crosslinked to a synthesized DNA strand, yielding a hOGG1-DNA complex. This complex was then crystallized and the diffraction data analyzed, yielding the crystal structure of the complex.


  1. Lindahl, T. Instability and decay of the primary structure of DNA. Nature 362, 709-715 (1993).
  2. Friedberg, E. C.  DNA damage and repair. Nature 421, 436–440 (2003).
  3. Lindahl, T. & Wood, R.D. Quality control by DNA repair. Science 286, 1897-1905 (1993)
  4. Neeley, W. L. & Essigmann, J. M. Mechanisms of formation, genotoxicity, and mutation of guanine oxidation products. Chem. Res. Toxicol. 19, 491–505 (2006).
  5. Mutsuo Sekiguchi and Teruhisa Tsuzuki, Oxidative nucleotide damage: consequences and prevention. Oncogene (2002) 21, 8895-8904
  6. Henle, E. S., and S. Linn. 1997. Formation, prevention, and repair of DNA damage by iron/hydrogen peroxide. J. Biol. Chem. 272:19095-19098.
  7. Bjelland S, Seeberg E. Mutagenicity, toxicity and repair of DNA base damage induced by oxidation. Mutat Res 2003;29:37-80.
  8. Oda Y et. al. NMR studies of a DNA containing 8-hydroxydeoxyguanosine. Nucleic Acids Res. 1991 Apr 11;19(7):1407–1412.
  9. Hsu, G. W., Ober, M., Carell, T. & Beese, L. S. Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature 431, 217–221 (2004).
  10. Shibutani, S., Takeshita, M. & Grollman, A. P. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349, 431–434
  11. Crenshaw, C. M., Structures and Dynamics of DNA Repair Enzymes: Mechanistic Insights into Catalysis and Damage Targeting [Thesis], Cambridge MA, Harvard University, 2009, 142 pp.
  12. S. David, V. O’Shea, and S. Kundu, Nature 447, 941-950 (2007)
  13. David, S. S. & Williams, S. D. Chemistry of glycosylases and endonucleases involved in base-excision repair. Chem. Rev. 98, 1221–1261 (1998).
  14. Boiteux S and Radicella JP (2001) Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step.  Nucleic Acids Research, 2001, Vol. 29, No. 6 1285-1292
  15. Bruner, S. D.; Norman, D. P. G.; Verdine, G. L. “Structural Basis for Recognition and Repair of the Endogenous Mutagen 8-oxoguanine in DNA,” Nature 2000, 403, 859-866.
  16. Dodson, M.L., Michaels, M.L. & Lloyd, R.S. Unified catalytic mechanism for DNA glycosylases. J. Biol. Chem. 269, 32709-32712 (1994).
  17. Vidal A.E., Hickson,I.D., Boiteux,S. and Radicella,J.P. Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step. Nucleic Acids Research, 2001, Vol. 29, No. 6 1285-1292
  18. Sheehan AM, McGregor DK, Patel A, Shidham V, Fan CY, Chang CC. Expression of human 8-oxoguanine DNA glycosylase (hOGG1) in follicular lymphoma. Modern Pathology (2005) 18, 1512–1518.
  19. Fan CY et. al. Frequent Allelic Imbalance and Loss of Protein Expression of the DNA Repair Gene hOGG1 in Head and Neck Squamous Cell Carcinoma.  Lab Invest 2001, 81:1429–1438
  20. O. Minowa, et. al., Mmh/Ogg1 gene inactivation results in accumulation of 8-hydroxyguanine in mice, Proc. Natl. Acad. Sci. U.S.A. 97 (2000), pp. 4156–4161
  21. A. Klungland, et. al., Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage, Proc. Natl. Acad. Sci. U.S.A. 96 (1999), pp. 13300–13305.
  22. K. Sakumi, et. al. Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption, Cancer Res. 63 (2003), pp. 902–905.
  23. Blainey, P. C.; van Oijen, A. M.; Banerjee, A.; Verdine, G. L.; Xie, X. S. “A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA,” Proc. Natl. Acad. Sci. USA 2006, 103, 5752-5757.
  24. Bowman, B. R.; Lee, S.; Wang, S.; Verdine, G. L. “Structure of the E. coli DNA Glycosylase AlkA Bound to the Ends of Duplex DNA: A System for the Facile Structure Determination of Lesion-Containing DNA,” Structure 2008, 16, 1166-1174.
  25. Lipscomb LA, et. al., X-ray structure of a DNA decamer containing 7,8-dihydro-8-oxoguanine. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):719-23.
  26. Oda, Y., et. al. 1991. NMR studies of a DNA containing 8-hydroxydeoxyguanosine. Nucleic Acids Res. 19:1407-1412.
  27. Banerjee, A.; Santos, W. L.; Verdine, G. L. “Structure of a DNA Glycosylase Searching for Lesions,” Science 2006, 311, 1155-1157.
  28. Fromme, J. C.; Verdine, G. L. “Structural insights into lesion recognition and repair by the bacterial 8-oxoguanine DNA glycosylase MutM,” Nat. Struct. Biol. 2002, 9, 544-552.
  29. Banerjee, A.; Verdine, G. L. “A nucleobase lesion remodels the interaction of its normal neighbor in a DNA glycosylase complex,” Proc. Natl. Acad. Sci. USA 2006, 103, 15020-15025.
  30. Banerjee, A.; Yang, W.; Karplus, M.; Verdine, G. L. “Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA,” Nature 2005, 434, 612-618.
  31. Radom, C.; Banerjee, A.; Verdine, G.L. “Structural characterization of human 8-oxoguanine DNA glycosylase variants bearing active site mutations,” J. Biol. Chem. 2007, 282, 9182-9194.
  32. Norman, D. P. G.; Chung, S. J.; Verdine, G. L. “Structural and biochemical exploration of a critical amino acid in human 8-oxoguanine glycosylase,” Biochemistry 2003, 42, 1564-1572.
  33. Huang, H., Harrison, S. C. & Verdine, G. L. Trapping of a catalytic HIV reverse transcriptase template:primer complex through a disulfide bond. Chem. Biol. 7, 355−364 (2000)
  34. Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 1669−1675 (1998)
  35. Fromme, J. C.; Bruner, S. D.; Yang, W.; Karplus, M.; Verdine, G. L. “Product-assisted catalysis in base-excision DNA repair,” Nat. Struct. Biol. 2003, 10, 204-211.
  36. Radom, C.T., Structural and Biochemical Exploration of Human 8-Oxoguanine Glycosylase I, in Chemistry and Chemical Biology. 2006, Harvard University: Cambridge, MA. p. 172.
  37. Zharkov, D.O. et al. Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J. 21, 789-800 (2002).
  38. Gilboa, R. et al. Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J. Biol. Chem. 277, 19811-19816 (2002).
  39. Chung, S. J.; Verdine, G. L. “Structures of End Products Resulting from Lesion Processing by a DNA Glycoylase/lyase,” Chem. & Biol. 2004, 11, 1643-1649.
  40. Lee, S.; Radom, C. T.; Verdine, G. L. “Trapping and Structural Elucidation of a Very Advanced Intermediate in the Lesion-Extrusion Pathway of hOGG1,” J. Am. Chem. Soc. 2008, 130, 7784-7785.
  41. Rodgers, D.W. Cryocrystallography. Structure 2, 1135-1140 (1994).
  42. Henderson, R. Cryo-protection of protein crystals against radiation damage in electron and x-ray diffraction. Proc. R. Soc. Lond. [Biol] 241, 6-8 (1990).
  43. Petsko, G. & Ringe, D. Protein Structure and Function (Sinauer Associates, Sunderland, Massachusetts, USA, 2003).
  44. Kavli, B., et. al., Excision of. Cytosine and Thymine from DNA by Mutants of Human. Uracil-DNA Glycosylase, EMBO J., 1996, vol. 15, no. 13, pp. 3442–3447.
  45. Bennett MT, et. al. Specificity of human thymine DNA glycosylase depends on N-glycosidic bond stability. J. Am. Chem. Soc. (2006) 128:12510–12519.
  46. Z. Otwinowski and W. Minor, “Processing of X-ray Diffraction Data Collected in Oscillation Mode”, Methods in Enzymology, Volume 276: Macromolecular Crystallography, part A, p.307-326, 1997, C.W. Carter, Jr. & R. M. Sweet, Eds., Academic Press (New York).
  47. A.T. Brunger, et. al.,Crystallography & NMR System (CNS), A new software suite for macromolecular structure determination, Acta Cryst. D 54, 905-921(1998).
  48. A.T. Brunger, Version 1.2 of the Crystallography and NMR System, Nature Protocols 2, 2728-2733 (2007).
  49. Zbyszek Otwinowski, W.M., Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology, 1997. 276: p. 307-326.
  50. Read, R.J., Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. Sect. A, 1986. 42: p. 104.
  51. A. W. Schuettelkopf and D. M. F. van Aalten (2004). PRODRG – a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D60, 1355–1363.

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