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.     

Excision of sugar-phosphate products at apurinic/apyrimidinic sites by DNA deoxyribophosphodiesterase of Escherichia coli.

It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli. A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate. It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E. coli. The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM. It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione. The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases.     

Isolation from barley embryo of endonuclease specific for apurinic sites in DNA

The endonuclease activity specific for apurinic sites in DNA was detected in barley embryos. The enzyme was partially purified. It reveals high activity on partially depurinated DNA but low or nil activity on intact and alkylated DNA. The method used for the detection of enzyme activity was based on the changes in the sedimentation velocity of substrate DNA in neutral sucrose gradients with 80 % formamide.     

Mechanism of DNA strand nicking at apurinic/apyrimidinic sites by Escherichia coli [formamidopyrimidine]DNA glycosylase.

Escherichia coli [formamidopyrimidine]DNA glycosylase catalyses the nicking of both the phosphodiester bonds 3' and 5' of apurinic or apyrimidinic sites in DNA so that the base-free deoxyribose is replaced by a gap limited by 3'-phosphate and 5'-phosphate ends. The two nickings are not the results of hydrolytic processes; the [formamidopyrimidine]DNA glycosylase rather catalyses a beta-elimination reaction that is immediately followed by a delta-elimination. The enzyme is without action on a 3'-terminal base-free deoxyribose or on a 3'-terminal base-free unsaturated sugar produced by a beta-elimination reaction nicking the DNA strand 3' to an apurinic or apyrimidinic site.     

Relation between Escherichia coli endonucleases specific for apurinic sites in DNA and exonuclease III.

Contradictory data have recently been published from two different laboratories on the presence vs absence of an intrinsic endonucliolytic activity of E. coli exonuclease III at apurinic sites in double-stranded DNA. It is shown here that an endonuclease activity of this specificity co-chromatographs exactly with exonuclease III on phosphocellulose and Sephadex G-75 columns, indicating that the endonuclease and exonuclease activities are due to the same enzyme. In addition, another E. coli endonuclease specific for apurinic sites exists, which can be separated from exonuclease III by the same chromatographic procedures.     

Homogeneous Escherichia coli FPG protein. A DNA glycosylase which excises imidazole ring-opened purines and nicks DNA at apurinic/apyrimidinic sites.

The repair of 2,6-diamino-4-hydroxy-5-N-methyl-formamidopyrimidine (Fapy) residues in DNA is performed by a Fapy-DNA glycosylase activity which is encoded for by the fpg gene in Escherichia coli. Besides DNA glycosylase activity, this protein, the FPG protein, is endowed with an EDTA-resistant activity nicking DNA at apurinic/apyrimidinic (AP) sites. To overproduce the FPG protein, the fpg gene was placed under the control of the tac promoter in the expression vector pKK223-3 yielding the pFPG230 plasmid. The production of the FPG protein in cells harboring the pFPG230 plasmid was 800-fold higher than that of the wild type strain after induction by isopropyl-beta-D-thio-galactopyranoside. From these cells, the FPG protein was purified to homogeneity in sufficient quantity to study its physical and catalytic properties. In its active form, the FPG protein is a globular monomer of 31 kDa and has an experimentally measured isoelectric point of 8.5. When the FPG protein is heat-denatured in the presence of EDTA the two activities are more rapidly inactivated than when heated in the absence of EDTA, suggesting that the FPG protein possesses a tightly bound metal ion. Atomic absorption spectrophotometric analysis shows that there is one zinc/FPG protein molecule. The FPG protein is different from previously described DNA glycosylases and AP-nicking enzymes in E. coli. The contribution of the AP-nicking activity associated with the FPG protein represents 10-20% of the total EDTA-resistant AP-nicking activities in E. coli.     

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.     

Mitochondrial endonuclease activities specific for apurinic/apyrimidinic sites in DNA from mouse cells

Endonuclease activity which specifically cleaves baseless (apurinic/apyrimidinic (AP] sites in supercoiled DNA has been purified from mitochondria of the mouse plasmacytoma cell line, MPC-11. Two variant forms separate upon purification; these have small but reproducible differences in catalytic and chromatographic properties, but similar physical properties. Both have a sedimentation coefficient of 4.0, corresponding to a molecular weight of 61,000 (assuming a globular configuration) and a peptide molecular weight of about 65,000 as determined by immunoblot analysis with antiserum raised against the major AP endonuclease from HeLa cells. Thus mitochondrial AP endonuclease appears to be a monomer of about 65 kDa, making it distinguishable from the major AP endonuclease of MPC-11 cells which, like those of other mammalian cells, appears to be a monomer of about 41 kDa. A possible 82-kDa precursor form was also detected by immunoblot analysis of a crude mitochondrial fraction. Mitochondrial AP endonuclease activity is greatly stimulated by divalent cations, has a pH optimum between 6.5 and 8.5, and cleaves the AP site by a class II mechanism to generate a 3'-OH nucleotide residue. These properties resemble those of the major mammalian AP endonucleases but, unlike those enzymes, mitochondrial AP endonuclease activity is neither inhibited by adenine or NAD+ nor stimulated by Triton X-100. Since the mitochondrial activity generates active primer termini for DNA synthesis, it could function in base excision DNA repair; alternatively, it might have a role in eliminating damaged mitochondrial genomes from the gene pool.     

A novel assay for endonucleases acting at apurinic sites and its use in measuring AP endonuclease activity in repair-deficient mutants of Saccharomyces cerevisiae.

A quick and convenient assay for depurination and AP endonuclease activities has been developed. (The term 'AP endonuclease' refers to a nuclease that acts on apurinic and probably apyrimidinic sites on DNA.) It is based on the observation that different topological forms of DNA, such as open circular DNA and covalently closed circular DNA, bind different amounts of the fluorescent intercalator ethidium bromide, and can therefore be distinguished by their fluorescence. This assay has been used to measure AP endonuclease activity in 22 repair-deficient mutants of Saccharomyces cerevisiae. All 22 had normal or nearly normal AP endonuclease activity. The AP endonuclease activity was partially characterized.     

Complementation of DNA repair-deficient Escherichia coli by the yeast Apn1 apurinic/apyrimidinic endonuclease gene

The Saccharomyces cerevisiae APN1 gene encoding an AP endonuclease/3'-diesterase was engineered in vitro for expression in Escherichia coli. The expression vector directs the synthesis in E. coli of a Mr 40,500 protein that reacts with anti-Apn1 antibodies and has the DNA-repair activities characteristic of Apn1 isolated from yeast. A band corresponding to Apn1 was observed in DNA repair activity gels only with extracts of E. coli harbouring the APN1 expression plasmid. Expression of Apn1 conferred resistance to oxidants and alkylating agents in E. coli lacking exonuclease III and endonuclease IV. For H2O2 damage, this rescue effect was correlated with the repair of oxidative lesions in the bacterial chromosome by the Apn1 protein. Thus, Apn1 can function in bacteria in a manner similar to its proposed multiple functions in yeast.     

Action of Escherichia coli and human 5'----3' exonuclease functions at incised apurinic/apyrimidinic sites in DNA.

The 5'----3' exonuclease activity of E. coli DNA polymerase I and a related enzyme activity in mammalian cell nuclei, DNase IV, are unable to catalyse the excision of free deoxyribose-phosphate from apurinic/apyrimidinic (AP) sites incised by an AP endonuclease. Instead, the sugar phosphate residue is slowly released as part of a short oligonucleotide. These products have been characterised as dimers and trimers by comparison of their retention time on reverse-phase HPLC with reference compounds prepared by acid depurination of a dinucleotide, trinucleotide and tetranucleotide containing a 5'-terminal dAMP residue. The similar mode of action of these enzymes at 5'-incised AP sites provides an explanation for the minority of repair patches larger than one nucleotide observed when AP sites are repaired by E. coli and mammalian cell extracts in vitro and strengthens the functional analogy between the two activities.     

Properties of an endonuclease activity in Micrococcus luteus acting on gamma-irradiated DNA and on apurinic DNA

A protein fraction from Micrococcus luteus with endonuclease activity against gamma-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against gamma-irradiated DNA was stimulated five-fold with 5 mM Mg++, whereas that against apurinic sites was less dependent on the Mg++ concentration. 100 mM KCl inhibited the gamma-ray endonuclease, but not the apurinic endonuclease activity. In gamma-irradiated RNA the protein recognized 1.65 endonuclease sensitive sites per radiation induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The affinity of the enzyme for the endonuclease sensitive site was evaluated resulting in a Km-value of 73 nM.     

Purification and properties of a Bacillus subtilis endonuclease specific for apurinic sites in DNA.

An endonuclease which hydrolyzes depurinated DNA has been purified from extracts of Bacillus subtilis cells. The endonuclease is a monomeric protein and has a molecular weight of around 56,000. The enzyme is specific for apurinic sites in double-stranded DNA, has a pH optimum at 8.0, and is slightly stimulated with 50 mM NaCl but completely inhibited with 500 mM NaCl. It requires no divalent cations and is insensitive to EDTA; it has no associated exonuclease. These properties are very similar to those of Escherichia coli endonuclease IV, which is also insensitive to EDTA and has no exonuclease activity, and very different from those of the main endonuclease for apurinic sites (endonuclease IV) of the same bacterium.     

Mapping of recognition sites for the restriction endonuclease from Escherichia coli K12 on bacteriophage PM2 DNA.

Several mutations in gene B of phage S13 appear to shorten the B protein by elimination of an N-terminal fragment, without destroying the B protein function. The shortened B protein resulting from each of these mutations can block the unique DNA-nicking properties of the S13 gene A protein. Because of the block in gene A function, normal gene B protein may have a function in phage DNA synthesis in addition to its known role in catalyzing capsid assembly.From gel electrophoresis the mutant B protein is estimated to be shorter than the normal S13 B protein by 1720 ± 70 daltons and is therefore believed to be an internal reinitiation fragment. The reinitiated fragments are functional and are made in about twice the amount of the normal B protein.The phage mutants which yield the reinitiation fragments are double mutants, each phage containing the same gene B nonsense mutation and each appearing to contain a different compensating gene B mutation. Various data support the assumption that the compensating mutations are frame-shifts, including the fact that suppression does not restore the normal-sized B protein. The reinitiation is assumed to occur at a pre-existing out-of-phase initiator codon, near the nonsense triplet; the correct reading frame would then be restored by each of the several different compensating mutations.The position of the normal S13 B protein in the gel electrophoresis pattern has been located both by elimination and shifting of the B peak, using appropriate amber mutants. The molecular weight of the S13 B protein is about 17,200, and is 2100 daltons less than the B protein of phage φX174; the S13 B protein can nevertheless substitute for the φX 174 B protein. Thus substantial portions of the B protein can be deleted without destroying its function.     

Human endonuclease specific for apurinic/apyrimidinic sites in DNA. Partial purification and characterization of multiple forms from placenta.

Six chromatographically distinct forms of endonuclease active on apurinic and apyrimidinic sites in DNA have been purified away from DNA phosphatases, DNA N-glycosidases, and other DNases of human placenta. The forms seem to be monomeric proteins of 27,000 to 31,000 daltons, and although catalytically similar, they can be distinguished from one another on the basis of substrate Km and the effects of small molecules such as ATP. Analysis of enzymatic activity on a spectrum of damaged DNA substrates indicates that the enzyme forms probably act at an appreciable rate only adjacent to the phosphodiester bond of a deoxyribose lacking a base (purine or pyrimidine) in duplex DNA; such sites can be formed by treating the DNA with acid, alkylating agents, DNA N-glycosidases, and, probably, x-rays and OsO4. The incision is made so as to form a deoxyribose 5'-phosphate and a 3'-hydroxynucleotide.     

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.     

A barley endonuclease specific for apurinic DNA. Isolation and partial characterization

An endonuclease specific for depurinated native DNA was isolated and partially purified from extracts of barley leaves. The procedure included streptomycin sulphate precipitation, ammonium sulphate fractionation, phosphocellulose, hydroxyapatite and Sephadex G-150 chromatography. Purity of the resulting enzyme was determined by gel electrophoresis and gel chromatography and specificity by testing the activity on intact and depurinated bacterial DNAs. At lower concentrations, the enzyme is specific for DNA containing apurinic sites. At higher concentrations, however, it degrades DNA in a non-specific manner. The nuclease has a pH optimum at 7.6, and a molecular weight of about 18000.     

Low concentrations of acridine dimers inhibit micrococcus AP endonuclease through interaction with apurinic sites in DNA.

The effect of dimeric DNA intercalating compounds was assayed on a purified AP endonuclease from Microccoccus luteus using apurinic supercoiled PM2 DNA as a substrate. Binding on apurinic sites was estimated through the competition with the intercalating compound, 9-NH2-ellipticine, which displays great specificity for apurinic sites. An acridine dimer with a spermine linker is at 0.1 microM the best inhibitor of cleavage at the apurinic site induced either by the AP endonuclease or by 9-NH2-ellipticine. Bisintercalating agents are more effective inhibitors of AP endonuclease than monointercalating ones. Most effective inhibitors among dimers have acridine residues.