The role of Mg2+ and specific amino acid residues in the catalytic reaction of the major human abasic endonuclease: new insights from EDTA-resistant incision of acyclic abasic site analogs and site-directed mutagenesis.

Ape1, the major protein responsible for excising apurinic/apyrimidinic (AP) sites from DNA, cleaves 5' to natural AP sites via a hydrolytic reaction involving Mg2+. We report here that while Ape1 incision of the AP site analog tetrahydrofuran (F-DNA) was approximately 7300-fold reduced in 4 mM EDTA relative to Mg2+, cleavage of ethane (E-DNA) and propane (P-DNA) acyclic abasic site analogs was only 20 and 30-fold lower, respectively, in EDTA compared to Mg2+. This finding suggests that the primary role of the metal ion is to promote a conformational change in the ring-containing abasic DNA, priming it for enzyme-mediated hydrolysis. Mutating the proposed metal-coordinating residue E96 to A or Q resulted in a approximately 600-fold reduced incision activity for both P and F-DNA in Mg2+compared to wild-type. These mutants, while retaining full binding activity for acyclic P-DNA, were unable to incise this substrate in EDTA, pointing to an alternative or an additional function for E96 besides Mg2+-coordination. Other residues proposed to be involved in metal coordination were mutated (D70A, D70R, D308A and D308S), but displayed a relatively minor loss of incision activity for F and P-DNA in Mg2+, indicating a non-essential function for these amino acid residues. Mutations at Y171 resulted in a 5000-fold reduced incision activity. A Y171H mutant was fourfold less active than a Y171F mutant, providing evidence that Y171 does not operate as the proton donor in catalysis and that the additional role of E96 may be in establishing the appropriate active site environment via a hydrogen-bonding network involving Y171. D210A and D210N mutant proteins exhibited a approximately 25,000-fold reduced incision activity, indicating a critical role for this residue in the catalytic reaction. A D210H mutant was 15 to 20-fold more active than the mutants D210A or D210N, establishing that D210 likely operates as the leaving group proton donor.     

MD simulation and experimental evidence for Mg²+ binding at the B site in human AP endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg(2+) dependent reaction. Despite characterization of nine X-ray crystallographic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg(2+) binding at the "A" site, we show experimentally that this mutant still requires Mg(2+) at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg(2+) placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg(2+) at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg(2+) binding at the B site is involved in the reaction mechanism associated with endonuclease function of APE1.     

Major oxidative products of cytosine are substrates for the nucleotide incision repair pathway

Most common point mutations occurring spontaneously or induced by ionizing radiation are C-->T transitions implicating cytosine as the target. Oxidative cytosine derivatives are the most abundant and mutagenic DNA damage induced by oxidative stress. Base excision repair (BER) pathway initiated by DNA glycosylases is thought to be the major pathway for the removal of these lesions. However, in alternative nucleotide incision repair (NIR) pathway the apurinic/apyrimidinic (AP) endonucleases incise DNA duplex 5' to an oxidatively damaged base in a DNA glycosylase-independent manner. Here, we characterized the substrate specificity of human major AP endonuclease, Ape1, towards 5-hydroxy-2'-deoxycytidine (5ohC) and alpha-anomeric 2'-deoxycytidine (alphadC) residues. The apparent kinetic parameters of the reactions suggest that Ape1 and the DNA glycosylases/AP lyases, hNth1 and hNeil1 repair 5ohC with a low efficiency. Nevertheless, due to the extremely high cellular concentration of Ape1, NIR was the major activity towards 5ohC in cell-free extracts. To address the physiological role of NIR function, we have characterized naturally occurring Ape1 variants including amino acids substitutions (E126A, E126D and D148E) and N-terminal truncated forms (NDelta31, NDelta35 and NDelta61). As expected, all Ape1 mutants had proficient AP endonuclease activity, however, truncated forms showed reduced NIR and 3'-->5' exonuclease activities indicating that these two functions are genetically linked and governed by the same amino acid residues. Furthermore, both Ape1-catalyzed NIR and 3'-->5' exonuclease activities generate a single-strand gap at the 5' side of a damaged base but not at an AP site in duplex DNA. We hypothesized that biochemical coupling of the nucleotide incision and exonuclease degradation may serve to remove clustered DNA damage. Our data suggest that NIR is a backup system for the BER pathway to remove oxidative damage to cytosines in vivo.     

A novel role of XRCC1 in the functions of a DNA polymerase beta variant

In the base excision repair pathway, wild-type DNA polymerase beta (WT polbeta) provides most of the gap filling synthesis. A truncated polbeta protein (polbetaDelta), expressed in primary colorectal and breast tumors and in a primary culture of renal cell carcinoma, inhibits the gap filling synthesis and DNA binding activities of WT polbeta. However, a purified recombinant polbetaDelta does not inhibit a purified WT polbeta. To determine the dominant inhibitory activity of polbetaDelta, we examined interactions of purified polbetaDelta with X-ray cross complementing group 1 (XRCC1), poly(ADP-ribose) polymerase (PARP), and apurinic endonuclease (Ape) proteins. All of these proteins interact with polbetaDelta in vitro and in vivo. The polbetaDelta protein can fill one nucleotide gap by inserting a base at the AP site, whereas a presumed binary complex of polbetaDelta and XRCC1 cannot. However, this binary complex not only suppresses gap filling synthesis activity of WT polbeta but also binds more strongly to gapped DNA than WT polbeta bound to XRCC1. These results are the first to suggest that XRCC1 is directly involved in the dominant negative activity of truncated polbeta, possibly leading to the genomic instability characteristic of tumor cells.     

Role of AP‐endonuclease (Ape1) active site residues in stabilization of the reactant enzyme‐DNA complex

Apurinic/apyrimidinic endonuclease 1 (Ape1) is an important metal‐dependent enzyme in the base excision repair mechanism, responsible for the backbone cleavage of abasic DNA through a phosphate hydrolysis reaction. Molecular dynamics simulations of Ape1 complexed to its substrate DNA performed for models containing 1 or 2 Mg²⁺‐ions as cofactor located at different positions show a complex with 1 metal ion bound on the leaving group site of the scissile phosphate to be the most likely reaction‐competent conformation. Active‐site residue His309 is found to be protonated based on pKa calculations and the higher conformational stability of the Ape1‐DNA substrate complex compared to scenarios with neutral His309. Simulations of the D210N mutant further support the prevalence of protonated His309 and strongly suggest Asp210 as the general base for proton acceptance by a nucleophilic water molecule.     

A recombinant exonuclease III homologue of the thermophilic archaeon Methanothermobacter thermautotrophicus

AP endonucleases catalyse an important step in the base excision repair (BER) pathway by incising the phosphodiester backbone of damaged DNA immediately 5' to an abasic site. Here, we report the cloning and expression of the 774 bp Mth0212 gene from the thermophilic archaeon Methanothermobacter thermautotrophicus, which codes for a putative AP endonuclease. The 30.3 kDa protein shares 30% sequence identity with exonuclease III (ExoIII) of Escherichia coli and 40% sequence identity with the human AP endonuclease Ape1. The gene was amplified from a culture sample and cloned into an expression vector. Using an E. coli host, the thermophilic protein could be produced and purified. Characterization of the enzymatic activity revealed strong binding and Mg2+-dependent nicking activity on undamaged double-stranded (ds) DNA at low ionic strength, even at temperatures below the optimum growth temperature of M. thermautotrophicus (65 degrees C). Additionally, a much faster nicking activity on AP site containing DNA was demonstrated. Unspecific incision of undamaged ds DNA was nearly inhibited at KCl concentration of approximately 0.5 M, whereas incision at AP sites was still complete at such salt concentrations. Nicked DNA was further degraded at temperatures above 50 degrees C, probably by an exonucleolytic activity of the enzyme, which was also found on recessed 3' ends of linearized ds DNA. The enzyme was active at temperatures up to 70 degrees C and, using circular dichroism spectroscopy, shown to denature at temperatures approaching 80 degrees C. Considering the high intracellular potassium ion concentration in M. thermautotrophicus, our results suggest that the characterized thermophilic enzyme acts as an AP endonuclease in vivo with similar activities as Ape1.     

Long-patch base excision DNA repair of 2-deoxyribonolactone prevents the formation of DNA-protein cross-links with DNA polymerase beta

Oxidized abasic sites are a major form of DNA damage induced by free radical attack and deoxyribose oxidation. 2-Deoxyribonolactone (dL) is a C1'-oxidized abasic site implicated in DNA strand breakage, mutagenesis, and formation of covalent DNA-protein cross-links (DPCs) with repair enzymes such as DNA polymerase beta (polbeta). We show here that mammalian cell-free extracts incubated with Ape1-incised dL substrates under non-repair conditions give rise to DPCs, with a major species dependent on the presence of polbeta. DPC formation was much less under repair than non-repair conditions, with extracts of either polbeta-proficient or -deficient cells. Partial base excision DNA repair (BER) reconstituted with purified enzymes demonstrated that Flap endonuclease 1 (FEN1) efficiently excises a displaced oligonucleotide containing a 5'-terminal dL residue, as would be produced during long-patch (multinucleotide) BER. Simultaneous monitoring of dL repair and dL-mediated DPC formation demonstrated that removal of the dL residue through the combined action of strand-displacement DNA synthesis by polbeta and excision by FEN1 markedly diminished DPC formation with the polymerase. Analysis of the patch size distribution associated with DNA repair synthesis in cell-free extracts showed that the processing of dL residues is associated with the synthesis of >or=2 nucleotides, compared with predominantly single nucleotide replacement for regular abasic sites. Our observations reveal a cellular repair process for dL lesions that avoids formation of DPCs that would threaten the integrity of DNA and perhaps cell viability.     

Human AP endonuclease possesses a significant activity as major 3'-5' exonuclease in human leukemia cells

Apurinic/apyrimidinic (AP) endonuclease (Ape1) is the major cellular enzyme responsible for repairing AP-sites in DNA. It can cleave the DNA phosphodiester backbone immediately 5(') to an AP-site. Ape1 also shows 3(')-phosphodiesterase activity, a 3(')-phosphatase activity, and an RNaseH activity. However, regarding its exonuclease activity, it remains controversial whether human Ape1 may possess a 3(')-5(') exonuclease activity. During the course of study to search for the major nuclease activity to double-stranded DNA in human leukemia cells, we purified a 37 kDa Mg(2+)-dependent exonuclease from cytosolic fraction of human leukemia U937 cells. Surprisingly, this exonuclease is Ape1. We demonstrated for the first time that Ape1 possesses a significant activity as major 3(')-5(') exonuclease in human leukemia cells. In addition, we also observed that translocation of cytoplasmic Ape1 into nucleus occurs during DNA damage.     

Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.     

Human apurinic/apyrimidinic endonuclease (Ape1) and its N-terminal truncated form (AN34) are involved in DNA fragmentation during apoptosis

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.     

Distinct roles of Ape1 protein in the repair of DNA damage induced by ionizing radiation or bleomycin

Ionizing radiation (IR) and bleomycin (BLM) are used to treat various types of cancers. Both agents generate cytotoxic double strand breaks (DSB) and abasic (apurinic/apyrimidinic (AP)) sites in DNA. The human AP endonuclease Ape1 acts on abasic or 3'-blocking DNA lesions such as those generated by IR or BLM. We examined the effect of siRNA-mediated Ape1 suppression on DNA repair and cellular resistance to IR or BLM in human B-lymphoblastoid TK6 cells and HCT116 colon tumor cells. Partial Ape1 deficiency (～30% of normal levels) sensitized cells more dramatically to BLM than to IR cytotoxicity. In both cases, expression of the unrelated yeast AP endonuclease, Apn1, largely restored resistance. Ape1 deficiency increased DNA AP site accumulation due to IR treatment but reduced the number of DSB. In contrast, for BLM, there were more DSB under Ape1 deficiency, with little change in the accumulation of AP sites. Although the role of Ape1 in generating DSB was greater for IR, the enzyme facilitated removal of AP sites, which may mitigate the cytotoxic effects of IR. In contrast, BLM generates scattered AP sites, and the DSB have 3'-phosphoglycolate termini that require Ape1 processing. These DSB persist under Ape1 deficiency. Apoptosis induced by BLM (but not by IR) under Ape1 deficiency was partially p53-dependent, more dramatically in TK6 than HCT116 cells. Thus, Ape1 suppression or inhibition may be a more efficacious adjuvant for BLM than for IR cancer therapy, particularly for tumors with a functional p53 pathway.     

Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.     

Impact of the C1' configuration of abasic sites on DNA duplex structure

Naturally occurring abasic sites in DNA exist as an equilibrium mixture of the aldehyde, the hydrated aldehyde, and the hemiacetal forms (dominant). The influence of the configuration of the C1' hydroxyl group of the hemiacetal form on duplex structure and abasic site repair has been examined using novel carbocyclic analogues. Both the alpha- and beta-forms of this novel abasic site were introduced into oligomeric DNA using the standard DMT-phosphoramidite approach in an automated solid-phase synthesizer. Solution structures of the d(CGTACXCATGC).d(GCATGAGTACG) duplex (where X is the alpha- or beta-anomer of the carbocyclic abasic site analogue) were determined by NMR spectroscopy and restrained molecular dynamics simulations. The structures were only minimally perturbed by the presence of either anomer of the abasic site. All residues adopted an anti conformation, and Watson-Crick alignments were observed on all base pairs of the duplexes. At the lesion site, the abasic residues and their partner adenines showed increased dynamic behavior but adopted intrahelical positions in the final refined structures. Incision of duplexes having the alpha- or beta-anomer of the carbocyclic abasic site by human AP endonuclease showed that the enzyme recognizes both configurations of the lesion and nicks the DNA backbone with similar efficiency. Our results challenge the suggestion that Ape1 is stereoselective and imply a plasticity at the active site of the enzyme for accommodating either anomer of the lesion.     

Functional characterization of Ape1 variants identified in the human population

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5′ to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited ~40–60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit ~10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility.     

Long patch base excision repair compensates for DNA polymerase β inactivation by the C4'-oxidized abasic site

The C4'-oxidized abasic site (C4-AP), which is produced by a variety of damaging agents, has significant consequences for DNA. The lesion is highly mutagenic and reactive, resulting in interstrand cross-links. The base excision repair of DNA containing independently generated C4-AP was examined. C4-AP is incised by Ape1 ~12-fold less efficiently than an apurinic/apyrimidinic lesion. DNA polymerase β induces the β-elimination of incised C4-AP in ternary complexes, duplexes, and single-stranded substrate. However, excision from a ternary complex is most rapid. In addition, the lesion inactivates the enzyme after approximately seven turnovers on average by reacting with one or more lysine residues in the lyase active site. Unlike 5'-(2-phosphoryl-1,4-dioxobutane), which very efficiently irreversibly inhibits DNA polymerase β, the lesion is readily removed by strand displacement synthesis conducted by the polymerase in conjunction with flap endonuclease 1. DNA repair inhibition by C4-AP may be a partial cause of the cytotoxicity of drugs that produce this lesion.     

Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinic endonuclease 1

The Ape1 protein initiates the repair of apurinic/apyrimidinic sites during mammalian base excision repair (BER) of DNA. Ape1 catalyzes hydrolysis of the 5'-phosphodiester bond of abasic DNA to create nicks flanked by 3'-hydroxyl and 5'-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) beta catalyzes both DNA synthesis at the 3'-hydroxyl terminus and excision of the 5'-dRP moiety prior to completion of BER by DNA ligase. During BER, Ape1 recruits pol beta to the incised apurinic/apyrimidinic site and stimulates 5'-dRP excision by pol beta. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Ape1 to modulate the deoxynucleotidyltransferase and 5'-dRP lyase activities of pol beta. We report here that Ape1 stimulates 5'-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Ape1, in which conditions that support DNA synthesis by pol beta also enhance 5'-dRP excision. Ape1 modulates the gap-filling activity of pol beta by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Ape1 protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol beta. However, this mutant protein retained the ability to stimulate 5'-dRP excision by pol beta. Simultaneous monitoring of 5'-dRP excision and DNA synthesis by pol beta demonstrated that the 5'-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER.