Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA

Several oxidative DNA-damaging agents, including ionizing radiation, can generate multiply damaged sites in DNA. Among the postulated lesions are those with abasic sites located in close proximity on opposite strands. The repair of an abasic site requires strand scission by a repair endonuclease such as human apurinic/apyrimidinic endonuclease (Ape) or exonuclease III in Escherichia coli. Therefore, a potential consequence of the "repair" of bistranded abasic sites is the formation of double-strand breaks. To test this possibility and to investigate the influence of the relative distance between the two abasic sites and their orientation to each other, we prepared a series of oligonucleotide duplexes containing abasic sites at defined positions either directly opposite each other or separated by 1, 3, or 5 base pairs in the 5'- or 3'-direction. Analysis following Ape and exonuclease III treatment of these substrates indicated a variety of responses. In general, cleavage at abasic sites was slower in duplexes with paired lesions than in control duplexes with single lesions. Double-strand breaks were, however, readily generated in duplexes with abasic sites positioned 3' to each other. With the duplex containing abasic sites set 1 base pair apart, 5' to each other, both Ape and exonuclease III slowly cleaved the abasic site on one strand only and were unable to incise the other strand. With the duplex containing abasic sites set 3 base pairs apart, 5' to each other, Ape protein was unable to cleave either strand. These data suggest that closely positioned abasic sites could have several deleterious consequences in the cell. In addition, this approach has allowed us to map bases that make significant contact with the enzymes when acting on an abasic site on the opposite strand.     

Excision of C-4'-oxidized deoxyribose lesions from double-stranded DNA by human apurinic/apyrimidinic endonuclease (Ape1 protein) and DNA polymerase beta

Oxidative damage to DNA deoxyribose generates oxidized abasic sites (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3'-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase beta, which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few-fold lower than incision of hydrolytic abasic sites at the same location. Consistent with several previous studies, Ape1 hydrolyzed 3'-phosphoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA polymerase beta excised the 5'-terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase beta-mediated excision of 5'-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coli Fpg (MutM) protein also excised 5'-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3'-phosphoglycolate esters.     

Rapid dissociation of human apurinic endonuclease (Ape1) from incised DNA induced by magnesium

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.     

3'-phosphodiesterase activity of human apurinic/apyrimidinic endonuclease at DNA double-strand break ends

In order to assess the possible role of human apurinic/apyrimidinic endonuclease (Ape) in double-strand break repair, the substrate specificity of this enzyme was investigated using short DNA duplexes and partial duplexes, each having a single 3'-phosphoglycolate terminus. Phosphoglycolate removal by Ape was detected as a shift in mobility of 5'-end-labeled DNA strands on polyacrylamide sequencing gels, and was quantified by phosphorimaging. Recombinant Ape efficiently removed phosphoglycolates from the 3'-terminus of an internal 1 base gap in a 38mer duplex, but acted more slowly on 3'-phosphoglycolates at a 19 base-recessed 3'-terminus, at an internal nick with no missing bases, and at a double-strand break end with either blunt or 2 base-recessed 3'-termini. There was no detectable activity of Ape toward 3'-phosphoglycolates on 1 or 2 base protruding single-stranded 3'-overhangs. The results suggest that both a single-base internal gap, and duplex DNA on each side of the gap are important binding/recognition determinants for Ape. While Ape may play a role in repair of terminally blocked double-strand breaks, there must also be additional factors involved in removal of at least some damaged 3'-termini, particularly those on 3'-overhangs.     

Expression of a putative catalytic domain of the human APEX nuclease (a major apurinic/apyrimidinic endonuclease) in Escherichia coli

1. Sequence analyses of APEX nuclease, a mammalian major apurinic/apyrimidinic (AP) endonuclease homologous to Escherichia coli exonuclease III, suggested that APEX nuclease is organized into two domains, a Mr 6000 N-terminal domain containing nuclear location signals and a Mr 29,000 C-terminal catalytic domain. 2. In order to study the enzyme structure further, vectors expressing APEX nuclease (pTAPXH1) and the Mr 29,000 C-terminal region (pTAPXH61) were constructed using cDNA (APX cDNA) for the human APEX nuclease and pTrc99A plasmid. The constructs were introduced into BW2001 strain (xth-11, nfo-2) cells of E. coli to produce transformants designated as BW2001/pTAPXH1 and BW2001/pTAPXH61, respectively. Both the APEX nuclease expressed in BW2001/pTAPXH1 and the Mr 29,000 C-terminal peptide expressed in BW2001/pTAPXH61 were partially purified by column chromatography and highly purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3. The purified APEX nuclease and the Mr 29,000 C-terminal peptide both showed equally high AP endonuclease activity which indicates that the Mr 29,000 C-terminal region of the APEX nuclease is (or contains) the AP endonuclease domain.     

Levels of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (APE1, APEX, Ref-1) are associated with the intrinsic radiosensitivity of cervical cancers

A study was made of the relationship between the intrinsic radiosensitivity of human cervical tumours and the expression of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both measurements were obtained on pretreatment biopsy material. All 25 tumours examined showed positive staining for HAP1, but there was heterogeneity in the level of expression both within and between tumours. The average coefficients of variation for intra- and intertumour heterogeneity were 62% and 82% respectively. There was a moderate but significant positive correlation between the levels of HAP1 expression and SF2 (r = 0.60, P = 0.002). Hence, this study shows that there is some relationship between intrinsic radiosensitivity and expression of a DNA repair enzyme in cervical carcinomas. The results suggest that this type of approach may be useful in the development of rapid predictive tests of tumour radiosensitivity.     

Differential cleavage of oligonucleotides containing the benzene-derived adduct, 1,N6-benzetheno-dA, by the major human AP endonuclease HAP1 and Escherichia coli exonuclease III and endonuclease IV

We report here that the newly synthesized DNA adduct, 1,N6-benzetheno-dA (pBQ-dA), in defined oligonucleotides [Chenna and Singer, Chem. Res. Toxicol., 8, 865-874], is a substrate for the major human AP endonuclease, HAP1, and the Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The mechanism of cleavage is identical to that reported previously for 3,N4-benzetheno-dC (pBQ-dC) and leads to a phosphodiester bond cleavage 5' to the adduct. There are, however, significant differences in the rate of cleavage of this adduct by these enzymes. The two bacterial AP endonucleases are both much more efficient than the human repair enzyme. In addition, using two random oligodeoxynucleotide sequences containing a single pBQ-dA, exonuclease III and endonuclease IV are similarly active, while HAP1 shows a distinct sequence preference of approximately 10-fold in efficiency of cleavage. The repair of this adduct by the three recombinant enzymes is further confirmed by using both active site mutant HAP1 proteins and by E.coli mutant strains lacking exonuclease III and/ or endonuclease IV. This sequence-dependent repair of pBQ-dA by HAP1 may play an important role in modulating benzene-induced carcinogenesis.     

Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI)

The Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA. To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence. We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol. Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site. In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site. These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site. The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site. The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.     

Normal processing of AP sites in Apn1-deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III

Escherichia coli exonuclease III and endonuclease III are two distinct DNA-repair enzymes that can cleave apurinic/apyrimidinic (AP) sites by different mechanisms. While the AP endonuclease activity of exonuclease III generates a 3'-hydroxyl group at AP sites, the AP lyase activity of endonuclease III produces a 3'-alpha,beta unsaturated aldehyde that prevents DNA-repair synthesis. Saccharomyces cerevisiae Apn1 is the major AP endonuclease/3'-diesterase that also produces a 3'-hydroxyl group at the AP site, but it is unrelated to either exonuclease III or endonuclease III. apn1 deletion mutants are unable to repair AP sites generated by the alkylating agent methyl methane sulphonate and display a spontaneous mutator phenotype. This work shows that either exonuclease III or endonuclease III can functionally replace yeast Apn1 in the repair of AP sites. Two conclusions can be derived from these findings. The first of these conclusions is that yeast cells can complete the repair of AP sites even though they are cleaved by AP lyase. This implies that AP lyase can contribute significantly to the repair of AP sites and that yeast cells have the ability to process the alpha,beta unsaturated aldehyde produced by endonuclease III. The second of these conclusions is that unrepaired AP sites are strictly the cause of the high spontaneous mutation rate in the apn1 deletion mutant.     

Cloning and characterization of a mouse homologue (mNthl1) of Escherichia coli endonuclease III

The association patterns between maternal anthropometric characteristics (stature, prepregnancy weight, prepregnancy body mass index, pregnancy weight gain) and newborn size (birth weight, length, head circumference) were tested with 10,240 single births taking place between 1985 and 1995 in Vienna, Austria, and 3,452 single births taking place between 1989 and 1995 in Westerstede-Ammerland (Friesland), northern Germany. Maternal size and newborn size differed highly significantly (p < 0.001) between the two genetically and socioeconomically different population groups. Furthermore, the incidence of macrosomia among newborns (birth weight greater than 4000 g) was extraordinarily high (17.9%) in the Frisian group from northern Germany. In both populations taller and heavier women with a higher weight gain during pregnancy gave birth to heavier offspring. Nevertheless, the pregnancy weight gain, which indicates environmental conditions of the mother, had only a minor impact on newborn size compared with stature and prepregnancy weight, which reflect the maternal genetic potential to a higher degree.     

Characterization of endonuclease III (nth) and endonuclease VIII (nei) mutants of Escherichia coli K-12

The nth and nei genes of Escherichia coli affect the production of endonuclease III and endonuclease VIII, respectively, glycosylases/apurinic lyases that attack DNA damaged by oxidizing agents. Here, we provide evidence that oxidative lethal lesions are repaired by both endonuclease III and endonuclease VIII and that spontaneous mutagenic lesions are repaired mainly by endonuclease III.     

Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.     

An unusual mechanism for the major human apurinic/apyrimidinic (AP) endonuclease involving 5' cleavage of DNA containing a benzene-derived exocyclic adduct in the absence of an AP site

The major human apurinic/apyrimidinic (AP) endonuclease (class II) is known to cleave DNA 5' adjacent to an AP site, which is probably the most common DNA damage produced hydrolytically or by glycosylase-mediated removal of modified bases. p-Benzoquinone (pBQ), one of the major benzene metabolites, reacts with DNA to form bulky exocyclic adducts. Herein we report that the human AP endonuclease directly catalyzes incision in a defined oligonucleotide containing 3,N4-benzetheno-2'-deoxycytidine (pBQ-dC) without prior generation of an AP site. The enzyme incises the oligonucleotide 5' to the adduct and generates 3'-hydroxyl and 5'-phosphoryl termini but leaves the pBQ-dC on the 5' terminus of the cleavage fragment. The AP function of the enzyme is not involved in this action, as no preexisting AP site is present nor is a DNA glycosylase activity involved. Nicking of the pBQ-dC adduct also leads to the same dangling base cleavage when two Escherichia coli enzymes, exonuclease III and endonuclease IV, are used. Our finding of this unusual mode of action used by both human and bacterial AP endonucleases raises important questions regarding the requirements for substrate recognition and catalytic active site(s) for this essential cellular repair enzyme. We believe this to be the first instance of the presence of a bulky carcinogen adduct leading to this unusual mode of action.     

Production of medium-chain-length poly(3-hydroxyalkanoates) from gluconate by recombinant Escherichia coli

Among the three kinds of the 2',3'-epoxypropyl beta-glycoside of disaccharides (GlcNAc-beta1,4-GlcNAc, Gal-beta1,4-GlcNAc, and Man-beta1,4-GlcNAc), the derivative of N-acetyllactosamine (Gal-beta1,4-GlcNAc-Epo) caused the dual labeling of human lysozyme (HL) most efficiently. The labeled HL was crystallized and analyzed by X-ray diffraction methodology. The X-ray analysis located the two Gal-beta1,4-GlcNAc-Epo moieties inside the catalytic cleft of HL. The attachment sites were the side-chain carboxylate groups of the catalytic residues Glu35 and Asp53 in HL. The first Gal-beta1, 4-GlcNAc-Epo moiety occupied virtually the same position as observed in the HL labeled with single Gal-beta1,4-GlcNAc-Epo molecule. The second Gal-beta1,4-GlcNAc-Epo moiety was recognized via the carbohydrate-carbohydrate interaction with the first Gal-beta1, 4-GlcNAc-Epo moiety in addition to the protein-carbohydrate interaction with the "right-side" catalytic cleft of HL through a number of hydrogen bonds including water-mediated ones as well as many van der Waals contacts. The two N-acetylglucosamine residues stacked with each other, while the two rings of galactose residues approximately shared the same plane. The dual labeling with two Gal-beta1,4-GlcNAc-Epo molecules was supposed to have occurred sequentially, which was accompanied with the alteration to the pKa of Glu35 derived from the esterification of Asp53 in the first labeling. Both asymmetric carbons in the connection parts between HL and N-acetyllactosamine moieties showed the same stereoconfiguration derived from the reaction with (2'R) stereoisomer concerning the epoxide group in the labeling reagent. The results demonstrated that the HL labeled with single Gal-beta1,4-GlcNAc-Epo was functional as a novel N-acetyllactosamine-binding protein, and the second labeling was performed by way of the first-ligand assisted recognition of the second ligand.     

Accessibility of epitopes on UvrB protein in intermediates generated during incision of UV-irradiated DNA by the Escherichia coli Uvr(A)BC endonuclease

Structural intermediates generated during incision of damaged DNA by the Uvr(A)BC endonuclease were probed with monoclonal antibodies (mAbs) raised against the Escherichia coli UvrB protein. It was found that the epitope of B2C5 mAb, mapped at amino acids (aa) 171-278 of UvrB, is not accessible in any of the preformed Uvr intermediates. Preformed B2C5-UvrB immunocomplexes, however, inhibited formation of those intermediates. B2C5 mAb seems to interfere with the formation of the UvrA-UvrB complex due to overlapping of its epitope and the UvrA binding region of UvrB. Conversely, the epitope of B3C1 mAb (aa 1-7 and/or 62-170) was accessible in all Uvr intermediates. The epitope of B*2E3 mAb (aa 171-278) was not accessible in any of the nucleoprotein intermediates preceding UvrB-DNA preincision complex. However, B*2E3 was able to immunoprecipitate this complex and to inhibit overall incision. B2A1 mAb (aa 8-61) inhibited formation of those Uvr intermediates requiring ATP binding and/or hydrolysis by UvrB. B*2B9 mAb (aa 473-630) inhibited Uvr nucleoprotein complexes involving UvrB. B*2B9 seems to prevent the binding of the UvrA-UvrB complex to DNA. The epitope of the B*3E11 mAb (aa 379-472) was not accessible in Uvr complexes formed at damaged sites. These results are discussed in terms of structure-functional mapping of UvrB protein.     

Low-level release of Shiga-like toxin (verocytotoxin) and endotoxin from enterohemorrhagic Escherichia coli treated with imipenem

Shiga-like toxin (SLT) and endotoxin may participate in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC) infection. Levels of release of SLT and endotoxin from EHEC treated in vitro with antibiotics were estimated. There were differential levels of release of SLT and endotoxin from EHEC treated with different antibiotics. Treatment of EHEC strains, namely, E. coli O157, O111, and O26, with imipenem induced much lower levels of release of SLT and endotoxin than treatment with ceftazidime.     

Stability and functionality of cysteine-less F(0)F1 ATP synthase from Escherichia coli

This case describes a new feature of fetal brain death syndrome, abnormal movements mimicking fetal convulsions being subsequently found to be decerebrate hypertonicity in a brain-dead fetus. It also confirms the diagnostic criteria of fetal brain death, both clinical and ultrasonic. The development of polyhydramnios both prior to and after the presumed neurological event is suggested as an association with the diagnosis of fetal brain death. Increased awareness of this event and the heterogeneity of the presentation may prevent further unnecessary Caesarean sections, as to date only 4 of the 10 cases in the literature were diagnosed prenatally. Utilization of techniques such as fetal blood sampling should be considered to further delineate the diagnosis.