Repair of clustered DNA lesions. Sequence-specific inhibition of long-patch base excision repair be 8-oxoguanine

Ionizing radiation induces clustered DNA damage where two or more lesions are located proximal to each other on the same or opposite DNA strands. It has been suggested that individual lesions within a cluster are removed sequentially and that the presence of a vicinal lesion(s) may affect the rate and fidelity of DNA repair. In this study, we addressed the question of how 8-oxoguanine located opposite to normal or reduced abasic sites would affect the repair of these sites by the base excision repair system. We have found that an 8-oxoguanine located opposite to an abasic site does not affect either the efficiency or fidelity of repair synthesis by DNA polymerase beta. In contrast, an 8-oxoguanine located one nucleotide 3'-downstream of the abasic site significantly reduces both strand displacement synthesis supported by DNA polymerase beta or delta and cleavage by flap endonuclease of the generated flap, thus inhibiting the long-patch base excision repair pathway.     

DNA damage levels and biochemical repair capacities associated with XRCC1 deficiency

Base excision repair (BER) is the major corrective pathway for most spontaneous, oxidative, and alkylation DNA base and sugar damage. X-ray cross-complementing 1 (XRCC1) has been suggested to function at nearly every step of this repair process, primarily through direct protein-protein interactions. Using whole cell extract (WCE) repair assays and DNA damage measurement techniques, we examined systematically the quantitative contribution of XRCC1 to specific biochemical steps of BER and single-strand break repair (SSBR). Our studies reveal that XRCC1-deficient Chinese hamster ovary WCEs exhibit normal base excision activity for 8-oxoguanine (8-OH-dG), 5-hydroxycytosine, ethenoadenine, and uracil lesions. Moreover, XRCC1 mutant EM9 cells possess steady-state levels of endogenous 8-OH-dG base damage similar to those of their wild-type counterparts. Abasic site incision activity was found to be normal in XRCC1-deficient cell extracts, as were the levels of abasic sites in isolated chromosomal DNA from mutant cells. While one- and five-nucleotide gap filling was not affected by XRCC1 status, a significant approximately 2-4-fold reduction in nick ligation activity was observed in EM9 WCEs. Our results herein suggest that the primary biochemical defect associated with XRCC1 deficiency is in the ligation step of BER/SSBR, and that XRCC1 plays no significant role in endogenous base damage and abasic site repair, or in promoting the polymerase gap-filling step.     

DNA Sequence Context Effects on the Glycosylase Activity of Human 8-Oxoguanine DNA Glycosylase

Human 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme involved in removing 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic DNA lesion generated by oxidative stress. The removal of 8-oxoG by OGG1 is affected by the local DNA sequence, and this feature most likely contributes to observed mutational hot spots in genomic DNA. To elucidate the influence of local DNA sequence on 8-oxoG excision activity of OGG1, we conducted steady-state, pre-steady-state, and single turnover kinetic evaluation of OGG1 in alternate DNA sequence contexts. The sequence context effect was studied for a mutational hot spot at a CpG dinucleotide. Altering either the global DNA sequence or the 5′-flanking unmodified base pair failed to influence the excision of 8-oxoG. Methylation of the cytosine 5′ to 8-oxoG also did not affect 8-oxoG excision. In contrast, a 5′-neighboring mismatch strongly decreased the rate of 8-oxoG base removal. Substituting the 5′-C in the CpG dinucleotide with T, A, or tetrahydrofuran (i.e. T:G, A:G, and tetrahydrofuran:G mispairs) resulted in a 10-, 13-, and 4-fold decrease in the rate constant for 8-oxoG excision, respectively. A greater loss in activity was observed when T:C or A:C was positioned 5′ of 8-oxoG (59- and 108-fold, respectively). These results indicate that neighboring structural abnormalities 5′ to 8-oxoG deter its repair thereby enhancing its mutagenic potential.     

Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis

Formamidopyrimidine-DNA glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion. Although the structure of Fpg has been established, amino acid residues that define damage recognition have not been identified. We have combined molecular dynamics and bioinformatics approaches to address this issue. Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions. On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O8 of extrahelical 8-oxoguanine accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis. Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and Arg-109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage. His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while Arg-108 provides specificity for bases positioned opposite the lesion.     

The yeast 8-oxoguanine DNA glycosylase (Ogg1) contains a DNA deoxyribophosphodiesterase (dRpase) activity.

The yeast OGG1 gene was recently cloned and shown to encode a protein that possesses N-glycosylase/AP lyase activities for the repair of oxidatively damaged DNA at sites of 7,8-dihydro-8-oxoguanine (8-oxoguanine). Similar activities have been identified for Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and Drosophila ribosomal protein S3. Both Fpg and S3 also contain a deoxyribophosphodiesterase (dRpase) activity that removes 2-deoxyribose-5-phosphate at an incised 5' apurinic/apyrimidinic (AP) sites via a beta-elimination reaction. Drosophila S3 also has an additional activity that removes trans-4-hydroxy-2-pentenal-5-phosphate at a 3' incised AP site by a Mg2+-dependent hydrolytic mechanism. In view of the substrate similarities between Ogg1, Fpg and S3 at the level of base excision repair, we examined whether Ogg1 also contains dRpase activities. A glutathione S-transferase fusion protein of Ogg1 was purified and subsequently found to efficiently remove sugar-phosphate residues at incised 5' AP sites. Activity was also detected for the Mg2+-dependent removal of trans -4-hydroxy-2-pentenal-5-phosphate at 3' incised AP sites and from intact AP sites. Previous studies have shown that DNA repair proteins that possess AP lyase activity leave an inefficient DNA terminus for subsequent DNA synthesis steps associated with base excision repair. However, the results presented here suggest that in the presence of MgCl2, Ogg1 can efficiently process 8-oxoguanine so as to leave a one nucleotide gap that can be readily filled in by a DNA polymerase, and importantly, does not therefore require additional enzymes to process trans -4-hydroxy-2-pentenal-5-phosphate left at a 3' terminus created by a beta-elimination catalyst.     

Effects of diethylmaleate on DNA damage and repair in the mouse brain

The enzyme 8-oxoguanine DNA glycosylase 1 participates in the repair of damaged DNA by excising the oxidized base 8-hydroxy-2'-deoxyguanosine. We have previously demonstrated that enzymatic activity of this enzyme is inversely related to the levels of the damaged base in specific brain regions. We now report that the activity of 8-oxoguanine DNA glycosylase 1 is increased in a region-specific manner following treatment with diethylmaleate, a compound that reduces glutathione levels in the cell. A single treatment with diethylmaleate elicited a significant increase ( approximately 2-fold) in the activity of 8-oxoguanine DNA glycosylase 1 in three brain regions with low basal levels of activity (cerebellum, cortex, and pons/medulla). There was no change in the activity of 8-oxoguanine DNA glycosylase 1 in those regions with high basal levels of activity (hippocampus, caudate/putamen, and midbrain). This is the first report to demonstrate that DNA repair capacity can be upregulated in the CNS, and the increased repair activity correlates with a reduction in the levels of DNA damage. The brain region-specific capacity to deal with increased oxidative damage to DNA may be responsible, in part, for the vulnerability of specific neuronal populations with aging, sources of oxidative stress, and neurodegenerative diseases.     

Repair of tandem base lesions in DNA by human cell extracts generates persisting single-strand breaks

Clustered DNA damage, where two or more lesions are located proximal to each other on the same or opposite DNA strands, is frequently produced as a result of exposure to ionising radiation. It has been suggested that such complex damaged sites pose problems for repair pathways. In this study, we addressed the question of how two 8-oxoguanine lesions, located two nucleotides apart on the same DNA strand, are repaired. We find that in human cell extracts repair of either of the 8-oxoguanine lesions within a tandem damaged site is initiated randomly and that the majority of the initiated repair proceeds to completion. However, a fraction of the initiated repair is delayed at the stage of an incised AP site and the rate of further processing of this incised AP site is dependent on the position of the remaining 8-oxoguanine. If the remaining 8-oxoguanine residue is located near the 5' terminus of the incised abasic site, repair continues as efficiently as repair of a single 8-oxoguanine residue. However, repair is delayed after the incision step when the remaining 8-oxoguanine residue is located near the 3' terminus. Although the presence of the 8-oxoguanine residue near the 3' terminus did not affect either DNA polymerase beta activity or poly(ADP)ribose polymerase-1 affinity and turnover on an incised AP site, we find that 8-oxoguanine-DNA glycosylase has reduced ability to remove an 8-oxoguanine residue located near the 3' terminus of the incised AP site. We find that binding of the 8-oxoguanine-DNA glycosylase to this 8-oxoguanine residue inhibits DNA repair synthesis by DNA polymerase beta, thus delaying repair. We propose that interference between a DNA glycosylase and DNA polymerase during the repair of tandem lesions may lead to accumulation of the intermediate products that contain persisting DNA strand breaks.     

Development of an enzymatic DNA repair assay for molecular epidemiology studies: distribution of OGG activity in healthy individuals

While the role of reduced DNA repair in susceptibility to hereditary cancers is well established, its role in sporadic cancer is less understood. One of the reasons is the lack of specific DNA repair assays that are suitable for epidemiology studies. Here we describe the development of the OGG test, an epidemiology-grade enzymatic assay for the activity of the base excision repair enzyme 8-oxoguanine DNA glycosylase, in protein extracts prepared from human blood cells. The assay is robust and reproducible, with a coefficient of variation of 10%. Using the OGG test we determined OGG activity in 120 healthy individuals. Our results show an inter-individual variation of 2.8-fold in OGG activity, from 3.6 up to 10.1units/microg protein, with a mean value of 7.2units/microg protein. There was no significant difference in OGG activity between males and females, or between smokers and non-smokers. Interestingly, there was a gender-specific effect of age: OGG activity was slightly but significantly lower in males older than the age of 55 years compared to younger males, but not in females at the same age groups. Analysis of OGG1 mRNA by quantitative real-time RT-PCR showed a group trend of an increase in OGG enzymatic activity with increasing mRNA expression, but the correlation between activity and mRNA in individuals was poor, indicating the importance of factors other than mRNA expression. The OGG test described is expected to be useful in studying the role of 8-oxoguanine repair in cancer, as recently demonstrated for non-small cell lung cancer [T. Paz-Elizur, M. Krupsky, S. Blumenstein, D. Elinger, E. Schechtman, Z. Livneh, J. Natl. Cancer Inst. 95 (2003) 1312-1319]. In addition, it may serve as a paradigm for the development of additional functional DNA repair tests, which are needed in order to gain further insight into the role of DNA repair in cancer risk and pathology.     

DNA repair as a biomarker in human biomonitoring studies; further applications of the comet assay

DNA repair plays a major role in maintaining genetic stability, and so measurement of individual DNA repair capacity should be a valued tool in molecular epidemiology studies. The comet assay (single cell gel electrophoresis), in different versions, is commonly used to measure the repair pathways represented by strand break rejoining, removal of 8-oxoguanine, and repair of bulky adducts or UV-induced damage. Repair enzyme activity generally does not reflect the level of gene expression; but there is evidence - albeit piecemeal - that it is affected by polymorphisms in repair genes. There are mixed reports concerning the regulation of repair by environmental factors; several nutritional supplementation trials with phytochemical-rich foods have demonstrated increases in base excision repair of oxidation damage, while others have shown no effect. Exposure to genotoxic agents has in general not been found to stimulate repair. Crucial questions concerning the factors regulating repair and the causes of individual variation are as yet unanswered.     

Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions

How DNA repair machineries detect and access, within the context of chromatin, lesions inducing little or no distortion of the DNA structure is a poorly understood process. Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway. We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin. The underlying mechanism does not require direct interaction of the protein with the oxidized base, however, the release of the protein from the chromatin fraction requires completion of repair. Inducing chromatin compaction by sucrose results in a complete but reversible inhibition of the in vivo repair of 8-oxoguanine. We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions.     

Base excision repair is impaired in mammalian cells lacking Poly(ADP-ribose) polymerase-1

In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.     

Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins

Clustered DNA lesions, possibly induced by ionizing radiation, constitute a trial for repair processes. Indeed, recent studies suggest that repair of such lesions may be compromised, potentially leading to the formation of lethal double-strand breaks (DSBs). A complex multiply damaged site (MDS) composed of 8-oxoguanine and 8-oxoadenine on one strand, 5-hydroxyuracil, 5-formyluracil and a 1 nt gap on the other strand, within 17 bp was built and used to challenge several steps of base excision repair (BER) pathway with human whole-cell extracts and purified repair enzymes as well. We show a hierarchy in the processing of lesions within the MDS, in particular at the base excision step. In the present configuration, efficient excision of 5-hydroxyuracil and low cleavage at 8-oxoguanine prevent DSB formation and generate a short single-stranded region carrying the 8-oxoguanine. On the other hand, rejoining of the 1 nt gap occurs by the short-patch BER pathway, but is slightly retarded by the presence of the oxidized bases. Taken together, our results suggest a hierarchy in the processing of the lesions within the MDS, which prevents the formation of DSB, but would dramatically enhance mutagenesis. They also indicate that the mutagenic (or lethal) consequences of a complex MDS will largely depend on the first event in the processing of the MDS.     

Phosphorylation of human oxoguanine DNA glycosylase (alpha-OGG1) modulates its function

Oxoguanine DNA glycosylase (OGG1) initiates the repair of 8-oxoguanine (8-oxoG), a major oxidative DNA base modification that has been directly implicated in cancer and aging. OGG1 functions in the base excision repair pathway, for which a molecular hand-off mechanism has been proposed. To date, only one functional and a few physical protein interactions have been reported for OGG1. Using the yeast two-hybrid system and a protein array membrane, we identified two novel protein interactions of OGG1, with two different protein kinases: Cdk4, a serine-threonine kinase, and c-Abl, a tyrosine kinase. We confirmed these interactions in vitro using recombinant proteins and in vivo by co-immunoprecipitation from whole cell extracts. OGG1 is phosphorylated in vitro by Cdk4, resulting in a 2.5-fold increase in the 8-oxoG/C incision activity of OGG1. C-Abl tyrosine phosphorylates OGG1 in vitro; however, this phosphorylation event does not affect OGG1 8-oxoG/C incision activity. These results provide the first evidence that a post-translational modification of OGG1 can affect its catalytic activity. The distinct functional outcomes from serine/threonine or tyrosine phosphorylation may indicate that activation of different signal transduction pathways modulate OGG1 activity in different ways.     

NEIL1 excises 3' end proximal oxidative DNA lesions resistant to cleavage by NTH1 and OGG1

Base excision repair is the major pathway for the repair of oxidative DNA damage in human cells that is initiated by a damage-specific DNA glycosylase. In human cells, the major DNA glycosylases for the excision of oxidative base damage are OGG1 and NTH1 that excise 8-oxoguanine and oxidative pyrimidines, respectively. We find that both enzymes have limited activity on DNA lesions located in the vicinity of the 3′ end of a DNA single-strand break, suggesting that other enzymes are involved in the processing of such lesions. In this study, we identify and characterize NEIL1 as a major DNA glycosylase that excises oxidative base damage located in close proximity to the 3′ end of a DNA single-strand break.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

The influence of formamidopyrimidine-DNA glycosylase on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZ alpha gene.

Base excision repair (BER) is a very important repair mechanism to cope with oxidative DNA damage. One of the most predominating oxidative DNA damages after exposure to ionizing radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This damage is repaired by formamidopyrimidine-DNA glycosylase (Fpg), a DNA glycosylase which is part of BER. Correct repair of 8oxoG is of great importance for cells, because 8oxoG has strong miscoding properties. Mispairing of 8oxoG with adenine instead of cytosine results in G:C to T:A transversion mutations. To determine the effect of a Fpg-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene, double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target, was irradiated with gamma-rays in aqueous solution under oxic conditions. Subsequently, the DNA was transfected into a wild-type Escherichia coli strain (JM105) and an isogenic Fpg-deficient E. coli strain (BH410). Although the overall spontaneous mutation spectra between JM105 and BH410 seemed similar, remarkable differences could be observed when the individual base pair substitutions were viewed. The amount of C to A transversions, which are most probably caused by unrepaired 8oxoG, has increased 3. 5-fold in the spontaneous BH410 spectrum. When the gamma-radiation-induced mutation spectra of JM105 and BH410 were compared, there was even a larger increase of C to A transversions in the BH410 strain (7-fold). We can therefore conclude that the straightforward approach used in this study confirms the importance of Fpg in repair of gamma-radiation-induced damage, and most probably especially in the repair of 8oxoG.     

极端嗜热古菌8oxoG DNA糖苷酶的研究进展

7,8二氢-8-氧鸟嘌呤(7,8-dihydro-8-oxoguanine,8oxoG)是一种常见的DNA损伤碱基.由于8oxoG能够与腺嘌呤配对,在DNA中的8oxoG被修复之前进行复制,DNA将会产生GC→TA的突变,从而造成基因组的不稳定.目前,碱基切除修复(Base excision repair,BER)是修...     

hMYH cell cycle-dependent expression, subcellular localization and association with replication foci: evidence suggesting replication-coupled repair of adenine:8-oxoguanine mispairs

The human MutY homolog, hMYH, is an adenine-specific DNA glycosylase that removes adenines or 2-hydroxyadenines mispaired with guanines or 8-oxoguanines. In order to prevent mutations, this activity must be directed to the newly synthesized strand and not the template strand during DNA synthesis. The subcellular localization and expression of hMYH has been studied in serum-stimulated, proliferating MRC5 cells. Using specific antibodies, we demonstrate that endogenous hMYH protein localized both to nuclei and mitochondria. hMYH in the nuclei is distinctly distributed and co-localized with BrdU at replication foci and with proliferating cell nuclear antigen (PCNA). The levels of hMYH in the nucleus increased 3- to 4-fold during progression of the cell cycle and reached maximum levels in S phase compared to early G₁. Similar results were obtained for PCNA, while there were no notable changes in expression of 8-oxoguanine glycosylase or the human MutT homolog, MTH1, throughout the cell cycle. The cell cycle-dependent expression and localization of hMYH at sites of DNA replication suggest a role for this glycosylase in immediate post-replication DNA base excision repair.     

Role of XRCC1 in the coordination and stimulation of oxidative DNA damage repair initiated by the DNA glycosylase hOGG1

XRCC1 participates in DNA single strand break and base excision repair (BER) to preserve genetic stability in mammalian cells. XRCC1 participation in these pathways is mediated by its interactions with several of the acting enzymes. Here, we report that XRCC1 interacts physically and functionally with hOGG1, the human DNA glycosylase that initiates the repair by BER of the mutagenic oxidized base 8-oxoguanine. This interaction leads to a 2- to 3-fold stimulation of the DNA glycosylase activity of hOGG1. XRCC1 stimulates the formation of the hOGG1 Schiff-base DNA intermediate without interfering with the endonuclease activity of APE1, the second enzyme in the pathway. On the contrary, the stimulation in the appearance of the incision product seems to reflect the addition of the effects of XRCC1 on the two first enzymes of the pathway. The data presented support a model by which XRCC1 will pass on the DNA intermediate from hOGG1 to the endonuclease APE1. This results in an acceleration of the overall repair process of oxidized purines to yield an APE1-cleaved abasic site, which can be used as a substrate by DNA polymerase beta. More importantly, the results unveil a highly coordinated mechanism by which XRCC1, through its multiple protein-protein interactions, extends its orchestrating role from the base excision step to the resealing of the repaired DNA strand.     

APE1-dependent repair of DNA single-strand breaks containing 3'-end 8-oxoguanine

DNA single-strand breaks containing 3′-8-oxoguanine (3′-8-oxoG) ends can arise as a consequence of ionizing radiation and as a result of DNA polymerase infidelity by misincorporation of 8-oxodGMP. In this study we examined the mechanism of repair of 3′-8-oxoG within a single-strand break using purified base excision repair enzymes and human whole cell extracts. We find that 3′-8-oxoG inhibits ligation by DNA ligase IIIα or DNA ligase I, inhibits extension by DNA polymerase β and that the lesion is resistant to excision by DNA glycosylases involved in the repair of oxidative lesions in human cells. However, we find that purified human AP-endonuclease 1 (APE1) is able to remove 3′-8-oxoG lesions. By fractionation of human whole cell extracts and immunoprecipitation of fractions containing 3′-8-oxoG excision activity, we further demonstrate that APE1 is the major activity involved in the repair of 3′-8-oxoG lesions in human cells and finally we reconstituted repair of the 3′-8-oxoG-containing oligonucleotide duplex with purified human enzymes including APE1, DNA polymerase β and DNA ligase IIIα.