Human Apurinic Endonuclease Research Articles

Radiation Resistance in Glioma Cells Determined by DNA Damage Repair Activity of Ape1/Ref-1

Since radiation therapy remains a primary treatment modality for gliomas, the radioresistance of glioma cells and targets to modify their radiation tolerance are of significant interest. Human apurinic endonuclease 1 (Ape1, Ref-1, APEX, HAP1, AP endo) is a multifunctional protein involved in base excision repair of DNA and a redox-dependent transcriptional co-activator. This study investigated whether there is a direct relationship between Ape1 and radioresistance in glioma cells, employing the human U87 and U251 cell lines. U87 is intrinsically more radioresistant than U251, which is partly attributable to more cycling U251 cells found in G2/M, the most radiosensitive cell stage, while more U87 cells are found in S and G1, the more radioresistant cell stages. But observed radioresistance is also related to Ape1 activity. U87 has higher levels of Ape1 than does U251, as assessed by Western blot and enzyme activity assays (approximately 1.5-2 fold higher in cycling cells, and approximately 10 fold higher at G2/M). A direct relationship was seen in cells transfected with CMV-Ape1 constructs; there was a dose-dependent relationship between increasing Ape1 overexpression and increasing radioresistance. Conversely, knock down by siRNA or by pharmacological down regulation of Ape1 resulted in decreased radioresistance. The inhibitors lucanthone and CRT004876 were employed, the former a thioxanthene previously under clinical evaluation as a radiosensitizer for brain tumors and the latter a more specific Ape1 inhibitor. These data suggest that Ape1 may be a useful target for modifying radiation tolerance.

Progress in the study of human apurinic endonuclease

各种致癌物和细胞毒性化合物均可引起DNA损伤.一些DNA损伤由太阳紫外线、烟草、不完全确定的饮食因素及环境毒物所引起,大部分DNA损伤则是由超氧化物自由基(ROS)和具有烷化作用的代谢物等引起.DNA修复系统在修复DNA损伤、维持DNA完整中起着不容忽视的作用,而碱基切除修复(BER)则是DNA修复系统中最主要的一种修复途径.在BER的第2步反应中,有一种脱嘌呤(AP)核酸内切酶参与,在人类细胞为人类脱嘌呤核酸内切酶1(HAP1,又称Apex、Ape1、Ref1),它是一种多功能蛋白质,可参与许多与细胞重要功能有关的反应.作为DNA修复酶,它作用于AP位点,是BER的重要酶类;作为氧化还原因子,它在转录因子活性降低时维持转录的进行,是维持细胞氧化还原状态的重要物质。

Human apurinic endonuclease 1 (APE1) expression and prognostic significance in osteosarcoma: Enhanced sensitivity of osteosarcoma to DNA damaging agents using silencing RNA APE1 expression inhibition

Osteosarcoma is the most common highly malignant bone tumor with primary appearance during the second and third decade of life. It is associated with a high risk of relapse, possibly resulting from a developed resistance to chemotherapy agents. As a means to overcome osteosarcoma tumor cell resistance and/or to sensitize tumor cells to currently used chemotherapeutic treatments, we examined the role of human apurinic endonuclease 1 (APE1) in osteosarcoma tumor cell resistance and prognosis. Sixty human samples of archived conventional (intramedullary) osteosarcoma were analyzed. APE1 protein was elevated in 72% of these tissues and among those with a known clinical outcome, there was a significant correlation between high APE1 expression levels and reduced survival times. The remaining 28% of samples showed low expression of APE1. Given that APE1 was overexpressed in osteosarcoma, we decreased APE1 levels using silencing RNA (siRNA) targeting technology in the osteosarcoma cell line, human osteogenic sarcoma (HOS), to enhance chemo- and radiation sensitivity. Using siRNA targeted technology of APE1, protein levels were reduced by more than 90% within 24 hours, remained low for 72 hours, and returned to normal levels at 96 hours. There was also a clear loss of APE1 endonuclease activity following APE1-siRNA treatment. A decrease in APE1 levels in siRNA-treated human osteogenic sarcoma cells led to enhanced cell sensitization to the DNA damaging agents: methyl methanesulfonate, H(2)O(2), ionizing radiation, and chemotherapeutic agents. The findings presented here have both prognostic and therapeutic implications for treating osteosarcoma. The APE1-siRNA results demonstrate the feasibility for the therapeutic modulation of APE1 using a variety of molecules and approaches.

Modulation of the 3′→5′-Exonuclease Activity of Human Apurinic Endonuclease (Ape1) by Its 5′-incised Abasic DNA Product

The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta.

Properties of and Substrate Determinants for the Exonuclease Activity of Human Apurinic Endonuclease Ape1

Ape1 is the major human abasic endonuclease, initiating repair of this common DNA lesion by incising the phosphodiester backbone 5′ to the damage site. This enzyme also functions in specific contexts to excise 3′-blocking termini, e.g. phosphate and phosphoglycolate residues, from DNA. Recently, the comparatively “minor” 3′ to 5′ exonuclease activity of Ape1 was found to contribute to the excision of certain 3′-mismatched nucleotides. In this study, I characterize more thoroughly the 3′-nuclease properties of Ape1 and define the effects of specific DNA determinants on this function. Data within shows that Ape1 is a non- or poorly processive exonuclease, which degrades one nucleotide gap, 3′-recessed, and nicked DNAs, but exhibits no detectable activity on blunt end or single-stranded DNA. A 5′-phosphate, compared to a 5′-hydroxyl group, reduced Ape1 degradation activity roughly tenfold, suggesting that the biological impact of certain DNA single strand breaks may be influenced by the terminal chemistry. In the context of a base excision repair-like DNA intermediate, a 5′-abasic residue exerted an about tenfold attenuation on the 3′ to 5′ exonuclease efficiency of Ape1. A 3′-phosphate group had little impact on Ape1 exonuclease activity, and oligonucleotides harboring these blocking termini were activated by Ape1 for DNA polymerase β extension. Ape1 was also found to remove 3′-tyrosyl residues from 3′-recessed and nicked DNAs, suggesting a potential role in processing covalent topoisomerase I–DNA intermediates formed during chromosome relaxation. While exhibiting preferential excision of thymine in a T:G mismatch context, Ape1 was unable to degrade a triple 3′-thymine mispair. However, Ape1 was able to excise double nucleotide mispairs, apparently through a novel 3′-flap-type endonuclease activity, again activating these substrates for polymerase β extension.

Action of human apurinic endonuclease (Ape1) on C1′-oxidized deoxyribose damage in DNA

Oxidative damage to DNA includes diverse lesions in the sugar-phosphate backbone. The chemical “nuclease” bis(1,10-phenanthroline)copper complex [(OP) 2Cu] is believed to generate a mixture of direct oxidative strand breaks and C1′-oxidized abasic sites (2-deoxyribonolactone; dL). We found that, under our conditions, the lesions produced by (OP) 2Cu (50 μM) in synthetic duplex DNA were predominantly dL, accompanied by ∼30% direct strand breaks with 3′-phosphates. For enzymatic studies, (OP) 2Cu was used to introduce damage with limited sequence-selectivity, while photolysis of a site-specific 2′-deoxyuridine-1′- t-butyl ketone generated dL at a defined position. The results showed that Ape1, the major human abasic endonuclease, catalyzed 5′-incision of dL sites, but acted at least 10-fold less effectively to remove the 3′-phosphates at direct strand breaks. Kinetic analysis of Ape1 incision using the site-specific dL substrate revealed the same k cat for dL and regular (glycosylase-generated) abasic sites, but with K m approximately five-fold higher for dL substrate. The efficiency of Ape1 acting on dL, and the abundance of this enzyme in vivo, indicate that dL sites in vivo would be rapidly processed by the endonuclease. The recent observation that Ape1-cleaved dL sites can covalently trap DNA polymerase β during the abasic excision process suggests that efficient incision of dL by Ape1 may potentiate further problems in DNA repair.

Urinary 8-oxo-2'-deoxyguanosine: redox regulation of DNA repair in vivo?

DNA is susceptible to damage by reactive oxygen species (ROS). ROS are produced during normal and pathophysiological processes in addition to ionizing radiation, environmental mutagens, and carcinogens. 8-oxo-2'-deoxyguanosine (8-oxodG) is probably one of the most abundant DNA lesion formed during oxidative stress. This potentially mutagenic lesion causes G --> T transversions and is therefore an important candidate lesion for repair, particularly in mammalian cells. Several pathways exist for the removal, or repair, of this lesion from mammalian DNA. The most established is via the base excision repair enzyme, human 8-oxoguanine glycosylase (hOgg1), which acts in combination with the human apurinic endonuclease (hApe). The latter is known to respond to regulation by redox reactions and may act in combination with hOgg1. We discuss evidence in this review article concerning alternative pathways in humans, such as nucleotide excision repair (NER), which could possibly remove the 8-oxodG lesion. We also propose that redox-active components of the diet, such as vitamin C, may promote such repair, affecting NER specifically.

Cellular radiosensitivity is not affected by mutated human apurinic endonuclease (hAPE)

Cellular radiosensitivity is not affected by mutated human apurinic endonuclease (hAPE)

Trypanosomal antioxidants and emerging aspects of redox regulation in the trypanosomatids.

Leishmania and Trypanosoma are two genera of the protozoal Order Kinetoplastida that cause widespread diseases of humans and their livestock. The production of reactive oxygen and nitrogen intermediates by the host plays an important role in the control of infections by these organisms. Signal transduction and its redox regulation have not been studied in any depth in trypanosomatids, but homologs of the redox-sensitive signal transduction machinery of other eukaryotes have been recognized. These include homologs of activator protein-1, human apurinic endonuclease 1 (Ref-1) endonuclease, iron-responsive protein, protein kinases, and phosphatases. The detoxification of peroxide is catalyzed by a trypanothione-dependent system that has no counterpart in mammals, and thus ranks as one of the biochemical peculiarities of trypanosomatids. There is substantial evidence that trypanothione is essential for the survival of Trypanosoma brucei and for the virulence of Leishmania spp. Apart from trypanothione and its precursors, trypanosomatids also possess significant amounts of N(1)-methyl-4-mercaptohistidine or ovothiol A, but its function in the trypanosomatids is not presently understood. The biosynthesis of ovothiol A in Crithidia fasciculata proceeds by addition of sulfur from cysteine to histidine to form 4-mercaptohistidine. S-(4'-L-Histidyl)-L-cysteine sulfoxide is the transsulfuration intermediate. 4-Mercaptohistidine is subsequently methylated with S-adenosylmethionine as the likely methyl donor.

Human Thymine DNA Glycosylase Binds to Apurinic Sites in DNA but Is Displaced by Human Apurinic Endonuclease 1

In vitro, following the removal of thymine from a G.T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S6-methylthioguanine (SMeG) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (koff 1.8 - 3.6 x 10(-5) s-1; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C.AP and SMeG.AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C.U and SMeG.T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G.AP sites sufficiently to allow it to remove more than one thymine from G.T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase beta, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site.

Dynamics of the Interaction of Human Apurinic Endonuclease (Ape1) with Its Substrate and Product

We investigated the interaction dynamics of human abasic endonuclease, the Ape1 protein (also called Ref1, Hap1, or Apex), with its DNA substrate and incised product using electrophoretic assays and site-specific amino acid substitutions. Changing aspartate 283 to alanine (D283A) left 10% residual activity, contrary to a previous report, but complementation of repair-deficient bacteria by the D283A Ape1 protein was consistent with its activity in vitro. The D308A, D283/D308A double mutant, and histidine 309 to asparagine proteins had 22, 1, and approximately 0. 02% of wild-type Ape1 activity, respectively. Despite this range of enzymatic activities, all the mutant proteins had near-wild-type binding affinity specific for DNA containing a synthetic abasic site. Thus, substrate recognition and cleavage are genetically separable steps. Both the wild-type and mutant Ape1 proteins bound strongly to the enzyme incision product, an incised abasic site, which suggested that Ape1 might exhibit product inhibition. The use of human DNA polymerase beta to increase Ape1 activity by eliminating the incision product supports this conclusion. Notably, the complexes of the D283A, D308A, and D283A/D308A double mutant proteins with both intact and incised abasic DNA were significantly more stable than complexes containing wild-type Ape1, which may contribute to the lower turnover numbers of the mutant enzymes. Wild-type Ape1 protein bound tightly to DNA containing a one-nucleotide gap but not to DNA with a nick, consistent with the proposal that substrate recognition by Ape1 involves a space bracketed by duplex DNA, rather than mere flexibility of the DNA.

Rapid Dissociation of Human Apurinic Endonuclease (Ape1) from Incised DNA Induced by Magnesium

Repair of apurinic/apyrimidinic (AP) sites is initiated by AP endonucleases, such as the human Ape1 protein (also called Hap1, Apex, and Ref1). This and related enzymes show strong dependence on divalent cations, particularly magnesium. Here we explore the role of this metal in different stages of the Ape1 reaction: substrate binding, cleavage, and product release. We examined DNA binding using an electrophoretic approach and DNA cleavage in single-turnover and steady-state reactions. Magnesium at low to moderate concentrations accelerated both substrate and product release by wild-type Ape1 protein. For a mutant Ape1 protein with an aspartate to alanine substitution at residue 308, substrate in preformed protein-DNA complexes was more efficiently cleaved before release in contrast to wild-type Ape1, whereas product release was accelerated dramatically. The magnesium dependence of steady-state AP endonuclease reactions was sigmoidal for both wild-type and the aspartate 308 to alanine protein but was not sigmoidal for an aspartate 283 to alanine derivative of Ape1. These results show that magnesium affects both DNA interactions with and phosphodiester cleavage by Ape1 and can change the rate-limiting step of the reaction. Structural studies will need to be interpreted in the context of these diverse effects of the metal.

Comparison of the promoters of the mouse (APEX) and human (APE) apurinic endonuclease genes

We investigated the minimal promoter of APEX, which encodes mouse apurinic DNA repair endonuclease. A 1.85-kb fragment with APEX upstream sequences and ∼290 bp of the transcribed region linked to a chloramphenicol acetyltransferase (CAT) reporter gene was assayed by transient transfection in NIH-3T3 cells. The minimal APEX promoter was comprised of ∼190 bp of upstream and ∼170 bp of transcribed DNA (exon 1 and most of intron 1). This ∼360-bp region contains two CCAAT boxes and other consensus protein binding sites, but no TATA box. Deletion of the 5′-most CCAAT box decreased activity ∼5-fold. The second CCAAT box (situated in exon 1) may play an independent role in APEX expression. Transcription start sites have been identified downstream of the second CCAAT box, and DNase I footprinting demonstrated NIH-3T3 nuclear proteins binding this region, including an Sp1 site located between the CCAAT boxes. Electrophoretic mobility-shift assays indicated binding by purified Sp1. Mouse proteins did not bind three myc-like (USF) sites in the APEX promoter, in contrast to the APE promoter. The APEX and APE promoter had similar activity in Hela cells, but in mouse cells, the murine promoter had ∼5-fold higher activity than did the human promoter. Both the APEX and APE promoters exhibited bidirectional activity in their cognate cells.

Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway.

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase beta (Polbeta) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein-protein contact between Polbeta and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Polbeta into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polbeta exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5'-terminal deoxyribose 5-phosphate by Polbeta. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.

Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites.

The mutagenic and lethal effects of abasic sites in DNA are averted by repair initiated by 'class II' apurinic (AP) endonucleases, which cleave immediately 5'to abasic sites. We examined substrate binding by the human AP endonuclease, Ape protein (also called Hap1, Apex or Ref-1). In electrophoretic mobility-shift experiments, Ape bound synthetic DNA substrates containing single AP sites or tetrahydrofuran (F) residues. No complexes were detected with single-stranded substrates or unmodified duplex DNA. In EDTA, the concentration of Ape required to shift 50% of duplex F-DNA was approximately 50 nM, while the addition of 10 mM MgCl2 nearly eliminated detectable F-DNA@Ape complexes. Filter-binding studies demonstrated a half-life of approximately 50 s at 0 degrees C for F-DNA@Ape complexes in the presence of EDTA, and <15 s after the addition of Mg2+. The DNA recovered from F-DNA@Ape complexes was intact but was rapidly cleaved upon addition of Mg2+, which suggests that these protein-DNA complexes are on the catalytic pathway for incision. Methylation and ethylation interference experiments identified DNA contacts critical for Ape binding, and Cu-1, 10-phenanthroline footprinting suggested an Ape-induced structural distortion at the abasic site prior to cleavage.

Characterization of the Promoter Region of the Human Apurinic Endonuclease Gene (APE)

Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5' to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites approximately 130 bp downstream of a putative "CCAAT box," approximately 130 bp 5' of the first splice junction in APE. Deletion of 5' sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promotor activity approximately 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region.

Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast.

Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis.

Biological responses of human apurinic endonuclease to radiation-induced DNA damage.

Biological responses of human apurinic endonuclease to radiation-induced DNA damage.

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert.

Reduction of Radiation Cytotoxicity by Human Apurinic Endonuclease in a Radiation-Sensitive Escherichia coli Mutant

Ionizing radiation produces a variety of DNA damage through active oxygen species such as the superoxide radical (O2.-), the hydroxyl radical (OH.), and hydrogen peroxide (H2O2). The removal of alkylation-induced apurinic (AP) sites and 3'-blocking deoxyribose fragments by exonuclease III (xth) and endonuclease IV (nfo) has been well demonstrated in E. coli. Very little information on the repair of radiation-induced DNA damage by human apurinic endonuclease is available. We examined the biological roles of the human AP endonuclease in the repair of radiation-induced DNA damage. An expression vector was constructed with human APE cDNA and transformed into radiation-sensitive E. coli mutants (xth- and nfo-). The radiation cytotoxicity was assayed by cell survival curves. Expression of human AP endonuclease in E. coli confirmed that AP endonuclease could complement exonuclease III functionally to diminish radiation cytotoxicity. In contrast, AP endonuclease was not able to increase resistance to H2O2, owing to a poor 3'-termini repair. We also tested whether AP endonuclease is a limiting factor for radiation cytotoxicity by using a plasmid nicking assay. Cell extracts from mutant cells with or without AP endonuclease expression were added to irradiated supercoiled plasmid DNA. The inability to convert supercoiled plasmid DNA to relaxed or linear forms suggested that there were large accumulations of AP sites in the mutant cell extracts. The AP endonuclease activities estimated from the plasmid nicking assays are 20-fold lower in the cell extracts of AP endonuclease-deficient mutant than in AP endonuclease-expressing cells. Therefore, AP endonuclease is a limiting step of base excision repair for the radiation-sensitive E. coli mutant, BW528. Our results conclude that AP endonuclease is responsible for the removal of AP sites from gamma-radiation-induced base damage in E. coli.