Comparison of apurinic DNA-binding protein from an ataxia telangiectasia and a HeLa cell line. Evidence for an altered processing of apurinic/apyrimidinic endonuclease

The major apurinic (AP) DNA-binding protein was purified from a HeLa cell line and from the SV40-transformed cell line AT5BIVA derived from a patient with the repair deficiency syndrome ataxia telangiectasia (AT). This protein appears to be identical with the major cellular apurinic/apyrimidinic endonuclease. The two endonucleases differ in their molecular weight (HeLa, 37,600; AT, 38,900) and their dissociation equilibrium constant for AP sites (HeLa, 7.8 X 10(-11) M; AT, 28.3 X 10(-11) M). These variances might be the consequence of a different post-translational modification. Evidence for this interpretation stems from the observation that the AP DNA binding activity of AP endonuclease, as measured in a glass-fiber filter binding assay, is inactivated upon incubation with snake venom phosphodiesterase and that the AP endonuclease from AT cells in 5-10-fold more sensitive than the HeLa enzyme. For both enzymes, the diesterase treatment leads to the formation of a protein of Mr 35,500 which might be the unmodified precursor of AP endonuclease. The loss of AP DNA binding does not reduce but rather increases the catalytic activity of AP endonuclease when measured at excess substrate concentration.     

Modification of deoxyribose-phosphate residues by extracts of ataxia telangiectasia cells

The release of DNA 5'-terminal deoxyribose-phosphate residues from enzymatically incised apurinic/apyrimidinic sites by human cell extracts has been under investigation. During the course of these studies, we observed that ataxia telangiectasia cell extracts modify deoxyribose-phosphate (dRp) residues by converting them to an altered form, dRp-X, which shows altered chromatographic properties on HPLC analysis. The chemical nature of the adduct is as yet unknown, but dRp-X is stable to both heat and acid. The modification requires an enzymatic activity and a low-molecular weight co-factor. Extracts of normal cells contain a dialyzable inhibitor that suppresses the reaction occurring with ataxia telangiectasia cell extracts. Formation of dRp-X has been observed in 7 out of 7 ataxia telangiectasia lymphoblastoid lines which represent at least 3 genetic complementation groups. Similar modification of dRp did not occur with extracts of cells of normal origin, nor those representing Fanconi's anaemia, xeroderma pigmentosum, Bloom's syndrome, Werner's syndrome or Friedreich's ataxia.     

Evaluation of the mouse mutant "wasted" as an animal model for ataxia telangiectasia. I. Age-dependent and tissue-specific effects

The wasted mouse, an animal model proposed for the genetically transmitted human disease ataxia telangiectasia (AT), was examined for its biological, cytogenetic and biochemical properties. In affected homozygotes, a marked age-dependent decrease in the ratio of spleen and thymus to body weight, and a slight but significant decrease in the liver to body weight ratio were observed while no such change was found in the kidney. An age-dependent increase was observed in the frequency of both spontaneous and gamma-ray-induced chromosomal aberrations in bone marrow cells of wasted mice. In littermate control mice, neither of these alterations was observed in an age-dependent manner. The activity of a primer activating enzyme, which has been reported to be deficient in AT cells, also decreased with age in spleen cells, but not in liver cells of affected mice. However, alterations in apurinic DNA endonuclease activity were not detected in the developmental stages examined. These data indicate that this mouse mutant may serve as a useful animal model for studying the relationships between DNA repair and lymphoid tissue differentiation.     

Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid.

A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.     

9-amino-ellipticine inhibits the apurinic site-dependent base excision-repair pathway.

The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base. This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E. coli through a mechanism involving apurinic sites. We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E. coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity. 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair. Other intercalating agents with similar affinities for DNA do not induce any inhibition. In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E. coli exonuclease III. The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity. 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration.     

Apurinic and/or apyrimidinic endonuclease activity in ataxia telangiectasia cell extracts.

The possibility that the increased sensitivity of ataxia telangiectasia towards ionizing radiation is related to a DNA-repair deficiency has been examined further. When compared to unaffected controls, 6 lines of fibroblast cells derived from ataxia patients demonstrated a slightly reduced endonucleolytic activity (165 +/- 12 units vs. 214 +/- 28 units) towards apurinic and/or apyrimidinic sites as determined in a "nicking" assay.     

Resistance to 6-thioguanine in spontaneously cycling and in mitogen-stimulated human peripheral lymphocytes

A study of the apurinic/apyrimidinic (AP) endonuclease activities of a mutant line of CHO cells, EM9, and its parental cell line, AA8, was undertaken to determine if the defective DNA repair exhibited by the mutant cell line after exposure to ethyl methanesulfonate was due to a defective AP endonuclease activity. Phosphocellulose chromatography of cell extracts resolved the AP endonuclease activities of both cell lines into two peaks as seen previously in mouse and human cells. No difference was found between the mutant and parental cell lines in the relative amount of AP endonuclease activity present in the two peaks.     

A highly conserved endonuclease activity present in Escherichia coli, bovine, and human cells recognizes oxidative DNA damage at sites of pyrimidines.

We have compared the sites of nucleotide incision on DNA damaged by oxidizing agents when cleavage is mediated by either Escherichia coli endonuclease III or an endonuclease present in bovine and human cells. E. coli endonuclease III, the bovine endonuclease isolated from calf thymus, and the human endonuclease partially purified from HeLa and CEM-C1 lymphoblastoid cells incised DNA damaged with osmium tetroxide, ionizing radiation, or high doses of UV light at sites of pyrimidines. For each damaging agent studied, regardless of whether the E. coli, bovine, or human endonuclease was used, the same sequence specificity of cleavage was observed. We detected this endonuclease activity in a variety of human fibroblasts derived from normal individuals as well as individuals with the DNA repair deficiency diseases ataxia telangiectasia and xeroderma pigmentosum. The highly conserved nature of such a DNA damage-specific endonuclease suggests that a common pathway exists in bacteria, humans, and other mammals for the reversal of certain types of oxidative DNA damage.     

Survival of apurinic SV40 DNA in the d-complementation group of xeroderma pigmentosum.

The survival of depurinated Form I SV40 DNA was studied in normal human fibroblasts and in D-complementation Xeroderma pigmentosum (XP) fibroblasts. Survival was measured with an infective center assay. Heat-acid and methyl methanesulfonate (MMS) were used as depurinating agents. After 3 hrs of depurination by heat--acid treatment, infectivity in normal cells was less than 15% of the controls compared to more than 50% for the XP D cell strains. Similar results were obtained with MMS-treated DNA. These results are contrary to expectation since apurinic endonuclease activity, which is presumed to be involved in the repair of apurinic sites, is much lower in XP D cell strains than in normal cell strains. Our results indicate that another mechanism for the repair of apurinic sites could exist.     

Properties of the main endonuclease specific for apurinic sites of Escherichia coli (endonuclease VI). Mechanism of apurinic site excision from DNA.

The main endonuclease for apurinic sites of Escherichia coli (endonuclease VI) has no action on normal strands, either in double-stranded or single-stranded DNA, or on alkylated sites. The enzyme has an optimum pH at 8.5, is inhibited by EDTA and needs Mg2+ for its activity; it has a half-life of 7 min at 40 degrees C. A purified preparation of endonuclease VI, free of endonuclease II activity, contained exonuclease III; the two activities (endonuclease VI and exonuclease III) copurified and were inactivated with the same half-lives at 40 degrees C. Endonuclease VI cuts the DNA strands on the 5' side of the apurinic sites giving a 3'-OH and a 5'-phosphate, and exonuclease III, working afterwards, leaves the apurinic site in the DNA molecule; this apurinic site can subsequently be removed by DNA polymerase I. The details of the excision of apurinic sites in vitro from DNA by endonuclease VI/exonuclease III, DNA polymerase I and ligase, are described; it is suggested that exonuclease III works as an antiligase to facilitate the DNA repair.     

A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci

The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.     

Residual ataxia telangiectasia mutated protein function in cells from ataxia telangiectasia patients, with 5762ins137 and 7271T-->G mutations, showing a less severe phenotype

We have assessed several ataxia Telangiectasia mutated (ATM)-dependent functions in cells derived from ataxia telangiectasia patients, carrying either an ATM 5762ins137 splice site or a 7271T-->G missense mutation, with a less severe phenotype compared with the classical disorder. ATM kinase in vitro, from 5762ins137 cells, showed the same specific activity as ATM in normal cells, but the protein was present at low levels. In contrast, mutant ATM kinase activity in the 7271T-->G cells was only about 6% that of the activity in normal cells, although the level of mutant protein expressed was similar to normal cells. Phosphorylation of the DNA double strand break repair proteins Nbs1 and hMre11, following DNA damage, was observed in normal and 7271T-->G cells but was almost absent in both 5762ins137 and classical ataxia telangiectasia cells. The kinetics of p53 response was intermediate between normal and classical ataxia telangiectasia cells in both the 7271T-->G and 5762ins137 cells, but interestingly, c-Jun kinase activation following DNA damage was equally deficient in cell lines derived from all the ataxia telangiectasia patients. Our results indicate that levels of ATM kinase activity, but not induction of p53 or c-Jun kinase activity, in these cells correlate with the degree of neurological disorder in the patients.     

Measurement of DNA polymerase beta in skin fibroblast cell lines from patients with ataxia telangiectasia

DNA polymerase beta levels were measured in 4 cell lines of normal human skin fibroblasts and in 5 cell lines of skin fibroblasts from patients with ataxia telangiectasia, an autosomal recessive disease exhibiting marked X-ray sensitivity. The enzyme specific activities for the normal lines were similar and the mean value was 2-fold lower than the mean value for the ataxia lines. With both kinds of cells, the enzyme level did not change as the cultures progressed from logarithmic to stationary phase of growth. Thus, this putative DNA repair enzyme appears to be 'constitutive' in human skin fibroblast lines, and a modest elevation of beta-polymerase activity is associated with ataxia telangiectasia. These results are discussed in the context to current views about DNA-repair enzymes in X-ray-sensitive cultured mammalian cells.     

Crystal structure and DNA repair activities of the AP endonuclease from Leishmania major

Apurinic/apyrimidinic endonucleases initiate the repair of abasic sites produced either spontaneously, from attack of bases by reactive oxygen species or as intermediates during base excision repair. The catalytic properties and crystal structure of Leishmania major apurinic/apyrimidinic endonuclease are described and compared with those of human APE1 and bacterial exonuclease III. The purified enzyme is shown to possess apurinic/apyrimidinic endonuclease activity of the same order as eukaryotic and prokaryotic counterparts and an equally robust 3'-phosphodiesterase activity. Consistent with this, expression of the L. major endonuclease confers resistance to both methyl methane sulphonate and H2O2 in Escherichia coli repair-deficient mutants while expression of the human homologue only reverts methyl methane sulphonate sensitivity. Structural analyses and modelling of the enzyme-DNA complex demonstrates a high degree of conservation to previously characterized homologues, although subtle differences in the active site geometry might account for the high 3'-phosphodiesterase activity. Our results confirm that the L. major's enzyme is a key element in mediating repair of apurinic/apyrimidinic sites and 3'-blocked termini and therefore must play an important role in the survival of kinetoplastid parasites after exposure to the highly oxidative environment within the host macrophage.     

Identification of mammalian DNA repair factors using a reconstituted subcellular system : Partial characterization and subcellular location of a DNA repair-stimulating protein in hamster cells

By reconstituting lysolecithin-permeabilized hamster cells with endogenous proteins, a protein(s) which stimulated bleomycin-induced DNA repair synthesis was identified. The repair protein was inactivated by proteinase K and had an apparent molecular weight of 12000–15000 D. The following enzymatic activities were not detected in the partially purified DNA repair protein: general endonuclease, apurinic endonuclease, exonuclease, DNA polymerase or DNA polymerase β-stimulating activity. The subcellular location of the DNA repair-stimulating activity was investigated by cytochalasin B enucleation; approx. 80% of the activity was associated with karyoplasts, suggesting a nuclear location. Neither the activity nor subcellular location of the repair protein fluctuated appreciably during the cell cycle, consistent with a physiological role in DNA repair. Although the function of the DNA repair protein is not yet known, this approach should be useful in identifying and characterizing mammalian DNA repair proteins.     

Apurinic endonuclease activity remains constant during early drosophila development

An endonuclease activity that acts on alkali-labile lesions in x-irradiated PM2 DNA and recognizes apurinic lesions in heat/acid treated DNA has been partially purified from Drosophila melanogaster embryos and its specific activity monitored throughout early development. The enzyme activity also showed a low level of activity on UV-irradiated DNA. The saturation kinetics observed with both x-irradiated and apurinic PM2 DNA substrates were similar. The endonuclease activity exhibited a broad pH optimum between pH 6 and 8.5 and was almost completely inhibited by 100 mM NaCl, 0.1 mM EDTA, 2 mM CaCl12 and 10 mM NEM. The reaction was not completely dependent on the presence of Mg⁺⁺cation, but optimum activity was obtained at a concentration of 0.1 mM; concentrations greater than 1 mM Mg^s++ were inhibitory. The specific activity of the apurinic endonuclease, partially purified from several stages of embryonic and early larval development, remained the same. Unfertilized eggs exhibited a reduced level of this presumptive repair activity.Abbreviations AP endonucleases Apurinic/apyrimidinic endonucleases     

DNA strained breakage repair in ataxia telangiectasia fibroblast-like cells.

Human diploid fibroblast-like cells derived from four patients with the genetic disease ataxia telangiectasia and from two non-mutant donors were examined for the repair of X-ray induced strand breaks in DNA. The ataxia telangiectasia cultures showed no significant differences from the non-mutant cultures in the kinetics and extent of strand repair. This suggests that the increased spontaneous and X-ray induced chromatid aberrations observed in ataxia telangiectasia cells are not caused by a defect in the repair of single strand breaks as might be suspected from a general model of aberration production.     

Heat shock protein 70 binds to human apurinic/apyrimidinic endonuclease and stimulates endonuclease activity at abasic sites

The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1 also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional consequence of the HSP70/HAP1 interaction was a 10-100-fold enhancement of endonuclease activity at abasic sites. The physical and functional interaction between HSP70 and HAP1 did not require the addition of ATP. The association of HSP70 and a key base excision repair enzyme suggests a role for heat shock proteins in promoting base excision repair. These findings provide a possible mechanism by which HSP70 protects cells against oxidative stress.     

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.     

Overexpression of human copper/zinc-superoxide dismutase in transgenic animals attenuates the reduction of apurinic/apyrimidinic endonuclease expression in neurons after in vitro ischemia and after transient global cerebral ischemia

Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Apurinic/apyrimidinic endonuclease (APE) is a multifunctional protein in the DNA base excision repair pathway which repairs apurinic/apyrimidinic sites in DNA. We investigated the involvement of oxidative stress and expression of APE in neurons after oxygen-glucose deprivation and after global cerebral ischemia. Our results suggest that overexpression of human copper/zinc-superoxide dismutase reduced oxidative stress with a subsequent decrease in APE expression. Production of oxygen free radicals and inhibition of the base excision repair pathway may play pivotal roles in the cell death pathway after ischemia.