Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding

HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells. Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme. Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity ~500-fold. Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1. These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1–D210E protein has a 3000-fold reduced K_cat for AP site cleavage, but an unchanged K_m. Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1–substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein. We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.     

Characterization of the AP endonucleases from Thermoplasma volcanium and Lactobacillus plantarum: Contributions of two important tryptophan residues to AP site recognition

Escherichia coli AP endonuclease (ExoIII) and its human homolog (APE1) have the sole tryptophan residue for AP site recognition (AP site recognizer) but these residues are at different positions near the catalytic sites. On the other hand, many bacterial AP endonucleases have two tryptophan residues at the same positions of both ExoIII and APE1. To elucidate whether these residues are involved in AP site recognition, the ExoIII homologs of Thermoplasma volcanium and Lactobacillus plantarum were characterized. These proteins showed AP endonuclease and 3'-5'exonculease activities. In each enzyme, the mutations of the tryptophan residues corresponding to Trp-280 of APE1 caused more significant reductions in activities and binding abilities to the oligonucleotide containing an AP site (AP-DNA) than those corresponding to Trp-212 of ExoIII. These results suggest that the tryptophan residue corresponding to Trp-280 of APE1 is the predominant AP site recognizer, and that corresponding to Trp-212 of ExoIII is the auxiliary recognizer.     

Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism

The mechanisms by which AP endonucleases recognize AP sites have not yet been determined. Based on our previous study with Escherichia coli exonuclease III (ExoIII), the ExoIII family AP endonucleases probably recognize the DNA-pocket formed at an AP site. The indole ring of a conserved tryptophan residue in the vicinity of the catalytic site presumably intercalates into this pocket. To test this hypothesis, we constructed a series of mutants of ExoIII and human APE1. Trp-212 of ExoIII and Trp-280 of APE1 were critical to the AP endonuclease activity and binding to DNA containing an AP site. To confirm the ability of the tryptophan residue to intercalate with the AP site, we examined the interaction between an oligopeptide containing a tryptophan residue and an oligonucleotide containing AP sites, using spectrofluorimetry and surface plasmon resonance (SPR) technology. The tryptophan residue of the oligopeptide specifically intercalated into an AP site of DNA. The tryptophan residue in the vicinity of the catalytic site of the ExoIII family AP endonucleases plays a key role in the recognition of AP sites.     

The role of Asn-212 in the catalytic mechanism of human endonuclease APE1: Stopped-flow kinetic study of incision activity on a natural AP site and a tetrahydrofuran analogue

Mammalian AP endonuclease 1 is a pivotal enzyme of the base excision repair pathway acting on apurinic/apyrimidinic sites. Previous structural and biochemical studies showed that the conserved Asn-212 residue is important for the enzymatic activity of APE1. Here, we report a comprehensive pre-steady-state kinetic analysis of two APE1 mutants, each containing amino acid substitutions at position 212, to ascertain the role of Asn-212 in individual steps of the APE1 catalytic mechanism. We applied the stopped-flow technique for detection of conformational transitions in the mutant proteins and DNA substrates during the catalytic cycle, using fluorophores that are sensitive to the micro-environment. Our data indicate that Asn-212 substitution by Asp reduces the rate of the incision step by ∼550-fold, while Ala substitution results in ∼70,000-fold decrease. Analysis of the binding steps revealed that both mutants continued to rapidly and efficiently bind to abasic DNA containing the natural AP site or its tetrahydrofuran analogue (F). Moreover, transient kinetic analysis showed that N212A APE1 possessed a higher binding rate and a higher affinity for specific substrates compared to N212D APE1. Molecular dynamics (MD) simulation revealed a significant dislocation of the key catalytic residues of both mutant proteins relative to wild-type APE1. The analysis of the model structure of N212D APE1 provides evidence for alternate hydrogen bonding between Asn-212 and Asp-210 residues, whereas N212A possesses an extended active site pocket due to Asn removal. Taken together, these biochemical and MD simulation results indicate that Asn-212 is essential for abasic DNA incision, but is not crucial for effective recognition/binding.     

Efficiency of incision of an AP site within clustered DNA damage by the major human AP endonuclease.

A major DNA lesion induced by ionizing radiation and formed on removal of oxidized base lesions by various glycosylases is an apurinic/apyrimidinic site (AP site). The presence of an AP site within clustered DNA damage, induced following exposure to ionizing radiation or radiomimetic anticancer agents, may present a challenge to the repair machinery of the cell, if the major human AP endonuclease, HAP1, does not efficiently incise the AP site. In this study, specific oligonucleotide constructs containing an AP site located at several positions opposite to another damage [5,6-dihydrothymine (DHT), 8-oxoG, AP site, or various types of single strand breaks] on the complementary strand were used to determine the relative efficiency of the purified HAP1 protein in incising an AP site(s) from clustered DNA damage. A base damage (DHT and 8-oxoG) on the opposite strand has little or no influence on the rate of incision of an AP site by HAP1. In contrast, the presence of either a second AP site or various types of single strand breaks, when located one or three bases 3' to the base opposite to the AP site, has a strong inhibitory effect on the efficiency of incision of an AP site. The efficiency of binding of HAP1 to an AP site is reduced by approximately 1 order of magnitude if a single strand break (SSB) is located one or three bases 3' to the site opposite to the AP site on the complementary strand. If the AP site and either a SSB or a second AP site are located at any of the other positions relative to each other, a double strand break may result.     

Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1.

A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries. This oligodeoxyribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation. HAP1 was found to recognize the strand break, and catalyze the release of the 3'-phosphoglycolate as free phosphoglycolic acid. The enzyme had a Vmax of 0.1 fmole/min/pg of HAP1 protein, and a Km of 0.05 microM for the 3'-phosphoglycolate strand break lesion. The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3'-phosphoglycolate moiety and the 3'-carbon of the adjacent dGMP moiety within the oligonucleotide. The resulting DNA contained a 3'-hydroxyl which supported nucleotide incorporation by E. coli DNA polymerase I large fragment. AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site. The Vmax and Km for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 microM, respectively.     

Abasic site recognition mechanism by the Escherichia coli exonuclease III.

In order to elucidate the mechanism of AP site recognition by Echerichia coli exonuclease III (exoIII), site-directed mutagenesis of the Tyr109, the Trp212, and the Phe213 residues, which were conserved in the type II AP endonuclease from various organisms and located in the vicinity of the catalytic site, was performed. The exoIII-W212S mutant lacked any detectable AP endonuclease activity and binding ability to the duplex DNA containing an AP site, while the exoIII-Y109S and exoIII-F213W mutants retained a low level of activities (13% and 83%, respectively, compared with wild-type exoIII). This study suggests that the Trp212 is an important component for abasic site recognition by the E. coli exonuclease III.     

Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity.

HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein.     

Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants.

Apurinic/apyrimidinic (AP) sites in cellular DNA are considered to be both cytotoxic and mutagenic, and can arise spontaneously or following exposure to DNA damaging agents. We have isolated cDNA clones which encode an endonuclease, designated HAP1 (human AP endonuclease 1), that catalyses the initial step in AP site repair in human cells. The predicted HAP1 protein has an Mr of 35,500 and shows striking sequence similarity (93% identity) to BAP 1, a bovine AP endonuclease enzyme. Significant sequence homology to two bacterial DNA repair enzymes, E. coli exonuclease III and S. pneumoniae ExoA proteins, and to Drosophila Rrp1 protein is also apparent. We have expressed the HAP1 cDNA in E. coli mutants lacking exonuclease III (xth), endonuclease IV (nfo), or both AP endonucleases. The HAP1 protein can substitute for exonuclease III, but not for endonuclease IV, in respect of some, but not all, DNA repair and mutagenesis functions. Moreover, a dut xth (ts) double mutant, which is nonviable at 42 degrees C due to an accumulation of unrepaired AP sites following excision of uracil from DNA, was rescued by expression of the HAP1 cDNA. These results indicate that AP endonucleases show remarkable conservation of both primary sequence and function. We would predict that the HAP1 protein is important in human cells for protection against the toxic and mutagenic effects of DNA damaging agents.     

The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites.

The structure of the major human apurinic/ apyrimidinic endonuclease (HAP1) has been solved at 2.2 A resolution. The enzyme consists of two symmetrically related domains of similar topology and has significant structural similarity to both bovine DNase I and its Escherichia coli homologue exonuclease III (EXOIII). A structural comparison of these enzymes reveals three loop regions specific to HAP1 and EXOIII. These loop regions apparently act in DNA abasic site (AP) recognition and cleavage since DNase I, which lacks these loops, correspondingly lacks AP site specificity. The HAP1 structure furthermore suggests a mechanism for AP site binding which involves the recognition of the deoxyribose moiety in an extrahelical conformation, rather than a 'flipped-out' base opposite the AP site.     

Pre-steady-state kinetic characterization of the AP endonuclease activity of human AP endonuclease 1

Human AP endonuclease 1 (APE1, REF1) functions within the base excision repair pathway by catalyzing the hydrolysis of the phosphodiester bond 5 ' to a baseless sugar (apurinic or apyrimidinic site). The AP endonuclease activity of this enzyme and two active site mutants were characterized using equilibrium binding and pre-steady-state kinetic techniques. Wild-type APE1 is a remarkably potent endonuclease and highly efficient enzyme. Incision 5 ' to AP sites is so fast that a maximal single-turnover rate could not be measured using rapid mixing/quench techniques and is at least 850 s(-1). The entire catalytic cycle is limited by a slow step that follows chemistry and generates a steady-state incision rate of about 2 s(-1). Site-directed mutation of His-309 to Asn and Asp-210 to Ala reduced the single turnover rate of incision 5 ' to AP sites by at least 5 orders of magnitude such that chemistry (or a step following DNA binding and preceding chemistry) and not a step following chemistry became rate-limiting. Our results suggest that the efficiency with which APE1 can process an AP site in vivo is limited by the rate at which it diffuses to the site and that a slow step after chemistry may prevent APE1 from leaving the site of damage before the next enzyme arrives to continue the repair process.     

Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo.

In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo. By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E. coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification. In addition MX-modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro. Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E. coli endonuclease III or IV in vitro.     

Three-dimensional structure prediction of bovine AP lyase, BAP1: prediction of interaction with DNA and alterations as a result of Arg176-->Ala, Asp282-->Ala, and His308-->Asn mutations

BAP1 is an apurinic/apyrimidinic lyase (AP lyase) that plays an important role in the repair of DNA damage. The present study deals with the prediction of the 3D structure of bovine AP lyase based on its sequence homology with human AP lyase. The predicted 3D model of bovine AP1 shows remarkable similarity with human endonuclease in the overall 3D fold. However, significant differences in the model and the X-ray structure were located at some of the important sites. We have analyzed the active center of the enzyme and other sites that are involved in DNA repair. A number of amino acids bind the bases located in the major/minor grooves of DNA. An insertion of Arg176 in the major groove and Met270 in the minor groove caps the DNA bound enzyme's active site, stabilizing the extra helical AP site conformation and effectively locking the protein onto the AP-DNA. Three BAP1 mutants were also modeled and analyzed as regards the changes in the structure. Substitution of Arg176-->Ala leads to the loss of DNA binding whereas mutation of Asp282-->Ala and His308-->Asn leads to a decrease in the enzymatic activity.     

Isolation of a small molecule inhibitor of DNA base excision repair

The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3'-phosphodiesterase and 3'-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy.     

8-OxoA inhibits the incision of an AP site by the DNA glycosylases Fpg, Nth and the AP endonuclease HAP1

Ionizing radiation induces clustered DNA damage sites, whereby two or more individual DNA lesions are formed within one or two helical turns of DNA by a single radiation track. A subset of DNA clustered damage sites exist in which the lesions are located in tandem on the same DNA strand. Recent studies have established that two closely opposed lesions impair the repair machinery of the cell, but few studies have investigated the processing of tandem lesions. In this study, synthetic double-stranded oligonucleotides were synthesized to contain 8-oxoA and an AP site in tandem, separated by up to four bases in either a 5' or 3' orientation. The influence 8-oxoA has on the incision of the AP site by the E. coli glycosylases Fpg and Nth protein and the human AP endonuclease HAP1 was assessed. 8-OxoA has little or no effect on the efficiency of incision of the AP site by Nth protein; however, the efficiency of incision of the AP site by Fpg protein is reduced in the presence of 8-oxoA even up to a four-base separation in both the 5' and 3' orientations. 8-OxoA influences the efficiency of HAP1 incision of the AP site only when it is 3' to the AP site and separated by up to two bases. This study demonstrates that the initial stages of base excision repair can be impaired by the presence of a second base lesion in proximity to an AP site on the same DNA strand. This impairment could have biological consequences, such as mutation induction, if the AP site is present at replication.     

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.     

Chlamydia pneumoniae AP endonuclease IV could cleave AP sites of double- and single-stranded DNA

Endonuclease IV gene, the only putative AP endonuclease of C. pneumoniae genome, was cloned into pET28a. Recombinant C. pneumoniae endonuclease I V (CpEndoIV) was expressed in E. coli and purified to homogeneity. CpEndoIV has endonuclease activity against apurinic/apyrimidinic sites (AP sites) of double-stranded (ds) oligonucleotides. AP endonuclease activity of CpEndoIV was promoted by divalent metal ions Mg2+ and Zn2+, and inhibited by EDTA. The natural (A, T, C and G) and modified (U, I and 8-oxo-G (GO)) bases opposite AP site had little effect on the cleavage efficiency of AP site of ds oligonucleotides by CpEndoIV. However, the CpEndoIV-dependent cleavage of AP site opposite modified base GO was strongly inhibited by Chlamydia DNA glycosylase MutY. Interestingly, the AP site in single-stranded (ss) oligonucleotides was also the effective substrate of CpEndoIV. Similar to E. coli endonuclease IV, AP endonuclease activity of CpEndoIV was also heat-stable to some extent, with a half time of 5 min at 60 degrees C.     

Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases.

Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates. Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein. Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases.