Human 8-Oxoguanine Glycosylase OGG1 Cleaves Abasic Sites and Covalently Conjugates to 3′-DNA Termini via Cysteine and Histidine Addition

Abstract

8-Oxoguanine glycosylase 1 (OGG1) repairs the major oxidative DNA damage, 8-oxo-2'-deoxyguanosine. It has been reported that OGG1 incises the most frequently formed DNA lesion, apurinic/apyrimidinic (AP) site, and in the process a stable DNA-OGG1 cross-link is formed. However, the chemical structure of the adduct is not characterized. Here, we report that DNA-OGG1 cross-links result from cysteine and histidine addition to incised AP sites at 3′-DNA termini.

Graphical Abstract

Human 8-Oxoguanine glycosylase 1 (OGG1) reacts with the most frequently generated DNA lesion, apurinic/apyrimidinic (AP) site, to form covalent DNA-protein cross-links (OGG1-DPCs). Using a battery of biochemical experiments and mass spectrometry, we determined that OGG1-DPCs result from cysteine and histidine addition to incised AP sites at 3′-DNA termini.

Introduction

8-Oxoguanine glycosylase 1 (OGG1) is a bifunctional DNA glycosylase that repairs 8-oxo-2'-deoxyguanosine, the major oxidative DNA damage.^[1] The apurinic/apyrimidinic (AP, Figure 1) site is one of the most abundant DNA damage.^[2] It is produced from spontaneous hydrolysis of N-glycosidic bonds in deoxyribonucleotides and induced by reactive oxygen species, DNA alkylating agents, ionizing radiation, and UV light.^[3] OGG1 binds tightly to AP sites.^[4] Prior studies by Lavrik and co-workers demonstrated that in vitro OGG1 reacts with an AP site and this reaction yields a covalent DNA-protein cross-link (DPC).^{[5, 6]} Different from other AP lyases, such as endonuclease III, that form transient DPCs, the OGG1-DPC can be detected by SDS-PAGE without prior stabilization by NaBH₄ reduction. Using a rapid approach to DNA adduct recovery assay in which DPCs were isolated followed by immunodetection, Demple and colleagues detected OGG1-DPC in MDA-MB-231 breast cancer cells that were treated with H₂O₂ or methyl methanesulfonate (MMS), a DNA methylating agent.^[7] DPCs are super-bulky DNA lesions and extremely toxic because they can block DNA replication and transcription.^[8] Accumulation of DPCs is linked to genome instability, aging, and cancer.^[9]

Figure 1.

Proposed 3′-PUA-OGG1 DPC structures and formation mechanisms.

Lavrik and co-workers found that mutating the catalytic Lys249 residue to glutamine abolished the OGG1-DPC formation.^[5] They therefore proposed that the NaBH₄-indpendent OGG1-DPC is linked through a Schiff base. Here, we wish to report evidence that supports distinct OGG1-DPC structures and propose a different DPC formation mechanism (Figure 1).

Results and Discussion

Lavrik and colleagues detected DPCs following reacting OGG1 with AP site-containing DNA in the absence of NaBH₄.^{[5, 6]} However, the DPC structure has not been determined. We reacted OGG1 with an AP site, isolated OGG1-DPC, and observed that the adduct slowly decomposed with a half-life of ~60 h at physiological temperature and pH (37 °C, pH 7.5, Figure S1). We also demonstrated that OGG1 cross-links to the incised AP site at 3'-DNA terminus as the proteinase K-treated OGG1-DPC migrated between the intact and heating-cleaved AP-DNA on a urea-PAGE gel (Figure S2). This observation can be explained by the formation of a Schiff base 3′-PUA-OGG1 DPC (Figure 1), which is supported by OGG1’s intrinsic AP lyase activity and that replacing the catalytic K249 residue by glutamine prevents the DPC formation.^{[4, 5]} However, the Schiff base can undergo hydrolysis to form 3'-phospho-α,β-unsaturated aldehyde (3'-PUA) that may conjugate to OGG1 through the Michael addition reaction (Figure 1). Alternatively, the nucleophilic addition could occur before hydrolysis. This type of cross-link has been observed for human mitochondrial transcription factor A, AlkB homolog 1, and poly(ADP-ribose) polymerase 1, in which Cys residues are the nucleophiles.^{[10, 11]} 3'-PUA can also react with glutathione that yields a similar product.^[12]

To determine whether the NaBH₄-independent OGG1-DPC is linked by a Schiff base or formed by Michael addition, we first conducted a gel-based experiment using an aldehyde-reactive probe (ARP, Figure 2a). If OGG1-DPC is linked by an imine, it either does not react with ARP, or decomposes to form 3′-PUA that can conjugate to ARP to yield 3′-PUA-ARP (Figure 2b). If OGG1 cross-links to DNA by Michael addition, the free aldehyde moiety will react with ARP via oxime ligation and such conjugation will not occur if the DPC is pre-reduced by NaBH₄ (Figure 2c). To explore that, the purified OGG1-DPC without or with prior NaBH₄ reduction was reacted with ARP, followed by proteinase K treatment and analysis by a urea-PAGE gel. 3′-PUA was produced by heating the AP site^[13] and reacted with ARP to serve as a control. As shown in Figure 2d, all non-reduced DPCs (Lanes 5-6), but not the reduced ones (Lanes 1-2), were converted to slower migrating products (i.e., proteolyzed 3′-PUA-OGG1-ARP). Notably, those ARP-conjugates also migrated more slowly than 3′-PUA-ARP (Figure 2d, Lanes 4 & 6). These data suggest that the OGG1-DPC has a free aldehyde moiety, which supports our proposed 3′-PUA-OGG1 DPC structure (Figure 1), in which a nucleophilic residue cross-links to C3′ before or after hydrolysis of the initially formed Schiff base 3′-PUA-OGG1 DPC intermediate.

Figure 2.

The 3′-PUA-OGG1 DPC has a free aldehyde. a). Structure of ARP. b). Possible reactions between Schiff base 3′-PUA-OGG1 DPC and ARP. c). Proposed reactions between Michael addition-induced 3′-PUA-OGG1 DPC and ARP. d). A 20% urea-PAGE gel analyzing the reactions of purified 3′-PUA-OGG1 DPCs and heated AP site with ARP. The gel was scanned using the fluorescence of 6-carboxyfluorescein. The two bands in lane 1 and two slower migrating bands in lane 5 were produced from incomplete degradation of the cross-linked OGG1 by proteinase K.

Furthermore, if OGG1 cross-links to the incised AP site via Michael addition, the DPC formation is expected to be inhibited by thiol-containing small molecules, such as 2-mercaptoethanol (β-ME, Figure 3a). Indeed, when OGG1 was incubated with the AP site in the presence of 1 mM β-ME, followed by SDS-PAGE analysis (Figure S3), the DPC yield decreased (Figure 3b). To verify that such reduction is exclusively caused by the competition of β-ME with OGG1, the reaction samples were digested by proteinase K and then analyzed by a urea-PAGE gel. As shown in Figure 3c-d, addition of β-ME did not affect the overall AP incision, but greatly inhibited the DPC formation.

Figure 3.

OGG1-DPC formation is inhibited by β-ME. a). A proposed mechanism that β-ME competes with OGG1 to conjugate to the incised AP site. b). DPC yields determined by SDS-PAGE. c-d). A representative 20% urea-PAGE gel and a plot showing amounts of remaining AP, proteolytic 3′-PUA-OGG1 DPC, and 3′-PUA/3′-PUA-β-ME. [AP] = 0.5 μM, [OGG1] = 5 μM, 37 °C, 7 h. The gel was scanned using the fluorescence of 6-carboxyfluorescein and # indicates a possible heat-labile DNA-DNA cross-link derived from the original AP site-containing DNA. The data in b) and d) are from three independent experiments and plotted as mean ± standard deviation. The p-values (***, p < 0.001, ****, p < 0.0001) were determined by two-tailed unpaired t test. n.s. = not significant.

Lastly, we identified the nucleophilic residues within OGG1 that conjugate to the incised AP site using liquid chromatography-tandem mass spectrometry (LC-MS/MS)^[10] (Figure 4a). This strategy utilizes tyrosyl-DNA phosphodiesterase 1 (TDP1), a 3′-PUA-DPC repair enzyme^{[10, 14]}, as a molecular tool to specifically cut the 3′-side of the phosphodiester bond and homogenously release OGG1 with a dynamic modification of C₅H₁₀O₃ (mass = 118.0630 Da). Specifically, the purified OGG1-DPC was reduced by NaBH₄ and the intact OGG1 was completely excised by excess TDP1 (Figure S4). The released protein was then alkylated by iodoacetamide, and digested by trypsin or endoproteinase GluC, followed by nano-LC-MS/MS analysis. In theory, nucleophilic side chains of Cys, Lys, and His residues can react with α,β-unsaturated aldehydes, such as 3′-PUA, through the Michael addition reaction.^[15] Therefore, the raw LC-MS/MS data were analyzed against the OGG1 amino acid sequence to identify peptides with the modification described above at a Cys, Lys, or His residue. We found four peptides that were modified at Cys28, Cys163, Cys241, and His270, respectively (Figure 4b-e). Based on a recently solved crystal structure of OGG1 and 2′-fluorine-8-OxoG-containing DNA^[1], the distances between these residues and C3′ are among 6.9 and 27.4 Å (Figure 4f), indicating that it is less likely that the Michael addition occurs before the hydrolysis of the Schiff base linkage between C1′ and Lys249. We therefore propose that OGG1 cross-links to 3′-PUA following hydrolysis of the initial Schiff base 3′-PUA-OGG1-DPC intermediate (Figure 1). To determine which residue is the major nucleophile, we individually mutated those four residues to serine or alanine, and then reacted that with the AP site and 3′-PUA, respectively. As shown in Figure S5, none of those mutants significantly reduced the DPC formation. This is possibly due to that other nucleophilic residues can compensate for one’s deficiency.

Figure 4.

Identification of OGG1 residues that cross-link to the incised AP site. a). Illustration of the LC-MS/MS pipeline. b-e). Mass spectra showing the identified OGG1 peptides with a mass adduct of 118.0630 Da at the cross-linking residue (*). f). Distances between OGG1 residues with C1′ or C3′ of deoxyribose connected to 2′-fluorine-8-OxoG, which is not shown for clarity. PDB #: 3ktu.

Conclusion

In conclusion, we provide evidence to support that OGG1 can cleave an AP site and then covalently conjugate to the 3′-DNA terminus via Cys or His addition to yield a reversible but long-lived DPC. We and others have previously demonstrated that 3′-PUA can react with cysteine residues to form 3′-PUA-DPCs.^{[10, 11]} To our knowledge, this is the first time to report the formation of a 3′-PUA-DPC via histidine addition.

It seems that the suicidal DPC formation acts against the role of OGG1 in base excision repair (BER). BER is a multistep process in which all repair enzymes are tightly coordinated to achieve efficient repair.^[16] We envision that under normal conditions, AP endonuclease 1 will rapidly repair AP sites and 3′-PUA^[17] and prevent the OGG1-DPC formation. However, when DNA damage is overwhelmed, such as upon the exposure to MMS or H₂O₂, AP sites and 3′-PUA will be accumulated^[18], which leads to the formation of OGG1-DPC. This notion is in line with the observation that OGG1-DPC is induced by those DNA-damaging agents in MDA-MB-231 breast cancer cells.^[7]