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.     

Differential reactivity of 9-NH2-ellipticine on apurinic and apyrimidinic sites in circular DNA

Endonucleases for apurinic sites as well as chemical compounds reacting with aldehydes do not generally differentiate between apurinic and apyrimidinic sites. We have studied the effect of the apurinic site reagent, 9-NH2-ellipticine, on apyrimidinic sites enzymatically generated on PBR322 DNA and compared it to its' action on apurinic PM2 and PBR322 DNAs. In conditions where this compound induces breakage of apurinic sites, it does not display any action on apyrimidinic sites.     

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.     

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.     

Mechanism of DNA cleavage and substrate recognition by a bovine apurinic endonuclease

The location of the phosphodiester bond cleaved by homogeneous Mg2+-dependent apurinic endodeoxyribonuclease (EC 3.1.25.2; APE) of bovine calf thymus has been determined by using a 21-mer oligonucleotide containing a single central apurinic site as a substrate. A single product of cleavage consistent with cleavage of the oligonucleotide 5' to the apurinic site, and leaving a 3' hydroxyl group, was identified. This enzyme is, therefore, a class II apurinic endonuclease. The substrate specificities of this enzyme have been determined by using a variety of natural and synthetic DNAs or oligonucleotides containing base-free sites. Calf thymus APE has an absolute requirement for a double-stranded DNA and requires an abasic site as a substrate. The presence of a base fragment such as a urea residue, an alkoxyamine group attached to the C'-1 position of the abasic site, or reduction of the C'-1 aldehyde abolishes the APE activity of this enzyme. Synthetic abasic sites containing either ethylene glycol, propanediol, or tetrahydrofuran interphosphate linkages are excellent substrates for bovine APE. These results indicate that APE has no absolute requirement for either ring-opened or ring-closed deoxyribose moieties in its recognition of DNA-cleavage substrates. The enzyme may interact with the pocket in duplex DNA that results from the base loss or with the altered conformations of the phosphodiester backbone that result from the abasic site.     

Interaction of poly(ADP-ribose) polymerase 1 with apurinic/apyrimidinic sites within clustered DNA damage

To study the interaction of poly(ADP-ribose) polymerase 1 (PARP1) with apurinic/apyrimidinic sites (AP sites) within clustered damages, DNA duplexes were created that contained an AP site in one strand and one of its analogs situated opposite the AP site in the complementary strand. Residues of 3-hydroxy-2-hydroxymethyltetrahydrofuran (THF), diethylene glycol (DEG), and decane-1,10-diol (DD) were used. It is shown for the first time that apurinic/apyrimidinic endonuclease 1 (APE1) cleaves the DNA strands at the positions of DEG and DD residues, and this suggests these groups as AP site analogs. Insertion of DEG and DD residues opposite an AP site decreased the rate of AP site hydrolysis by APE1 similarly to the effect of the THF residue, which is a well-known analog of the AP site, and this allowed us to use such AP DNAs to imitate DNA with particular types of clustered damages. PARP1, isolated and in cell extracts, efficiently interacted with AP DNA with analogs of AP sites producing a Schiff base. PARP1 competes with APE1 upon interaction with AP DNAs, decreasing the level of its cross-linking with AP DNA, and inhibits hydrolysis of AP sites within AP DNAs containing DEG and THF residues. Using glutaraldehyde as a linking agent, APE1 is shown to considerably decrease the amount of AP DNA-bound PARP1 dimer, which is the catalytically active form of this enzyme. Autopoly(ADP-ribosyl)ation of PARP1 decreased its inhibitory effect. The possible involvement of PARP1 and its automodification in the regulation of AP site processing within particular clustered damages is discussed.     

Mechanism of action of Micrococcus luteus gamma-endonuclease

Micrococcus luteus extracts contain gamma-endonuclease, a Mg2+-independent endonuclease that cleaves gamma-irradiated DNA. This enzyme has been purified approximately 1000-fold, and the purified enzyme was used to study its substrate specificity and mechanism of action. gamma-Endonuclease cleaves DNA containing either thymine glycols, urea residues, or apurinic sites but not undamaged DNA or DNA containing reduced apurinic sites. The enzyme has both N-glycosylase activity that releases thymine glycol residues from OsO4-treated DNA and an associated apurinic endonuclease activity. The location and nature of the cleavage site produced has been determined with DNA sequencing techniques. gamma-Endonuclease cleaves DNA containing thymine glycols or apurinic sites immediately 3' to the damaged or missing base. Cleavage results in a 5'-phosphate terminus and a 3' baseless sugar residue. Cleavage sites can be converted to primers for DNA polymerase I by subsequent treatment with Escherichia coli exonuclease III. The mechanism of action of gamma-endonuclease and its substrate specificity are very similar to those identified for E. coli endonuclease III.     

Neocarzinostatin-induced DNA base release accompanied by staggered oxidative cleavage of the complementary strand

Treatment of an end-labeled DNA restriction fragment with the nonprotein chromophore of neocarzinostatin induced lesions which, after treatment with endonuclease IV or putrescine, were expressed as site-specific double-strand breaks. Analysis of the termini at cleavage sites in each strand showed that the neocarzinostatin-induced lesions consisted of an apurinic/apyrimidinic site plus a closely opposed break in the complementary strand. The break always occurred opposite the base two positions upstream from the apurinic/apyrimidinic site and had the 3'-phosphate and 5'-aldehyde termini characteristic of neocarzinostatin-induced breaks. This positioning suggests that neocarzinostatin simultaneously attacks two DNA sugars on opposite edges of the minor groove. The sequence specificity for formation of apurinic/apyrimidinic sites with closely opposed breaks reflected that of neocarzinostatin-induced mutagenesis. The potent mutagenicity of these lesions may be attributable to the presence of closely opposed damage in both DNA strands.     

DNA containing a chemically reduced apurinic site is a high affinity ligand for the E. coli formamidopyrimidine-DNA glycosylase.

The E. coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities. The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA. Besides DNA glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites). In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein. However, the DNA glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative. The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels. The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride. The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis. Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined. In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site.     

Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.     

A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ

A new enzyme, which we named ribosomal RNA apurinic site specific lyase (RALyase), is described. The protein was found in wheat embryos and has a molecular weight of 50 625 Da. The enzyme specifically cleaves the phosphodiester bond at the 3' side of the apurinic site introduced by ribosome-inactivating proteins into the sarcin/ricin domain of 28S rRNA. The 3' and 5' ends of wheat 28S rRNA at the cleavage site are 5'-GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA-3', demonstrating that the enzyme catalyzes a beta-elimination reaction. The substrate specificity of the enzyme is extremely high: it acts only at the apurinic site in the sarcin/ricin domain of intact ribosomes, not on deproteinized rRNA or DNA containing apurinic sites. The amino acid sequences of five endopeptidase LysC-liberated peptides from the purified enzyme were determined and used to obtain a cDNA sequence. The open reading frame encodes a protein of 456 amino acids, and a homology search revealed a related rice protein. Similar enzyme activities were also found in other plants that express ribosome-inactivating proteins. We believe that RALyase is part of a complex self-defense mechanism.     

Replication of DNA containing apurinic sites in human and mouse cells probed with parvoviruses MVM and H-1.

We studied the effect of apurinic sites on DNA replication in mouse and human cells, using parvoviruses MVM (minute virus of mice) and H-1 as probes. Although apurinic sites are efficient blocks to the replication of these single-stranded DNA viruses in vivo, depurinated parvoviruses can be reactivated if host cells have been preexposed to a subtoxic dose of UV light. The target of this conditional reactivation process is the conversion of depurinated input DNA into double-stranded replicative forms; the concomitant increase in viral mutagenesis strongly suggests that apurinic sites can be bypassed in mammalian cells.     

The DNA bending by acetylaminofluorene residues and by apurinic sites

We have studied the distortions induced in double-stranded oligonucleotides by covalently bound acetylaminofluorene residues and by apurinic sites. Within the acetylaminofluorene-modified oligonucleotide three base-pairs are unpaired as detected by the chemical probes chloroacetaldehyde and osmium tetroxide. These two probes reveal that the bases adjacent to the apurinic site are paired. In both the modified double-stranded oligonucleotides, the backbone on the 5' side of the modification is more reactive with 1,10-phenanthroline copper than the backbone on the 3' side. On polyacrylamide gels, the ligated multimers of acetylaminofluorene or apurinic site-modified oligonucleotides migrate slower than the multimers of the unmodified oligonucleotides. It is suggested that the acetylaminofluorene-modified guanine residues and the apurinic sites behave more as hinge joints than as the centres of directed bends.     

Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but beta-elimination and sometimes beta delta-elimination catalysts.

Bacteriophage-T4 UV endonuclease nicks the C(3')-O-P bond 3' to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction. The breakage of this bond is sometimes followed by the nicking of the C(5')-O-P bond 5' to the AP site, leaving a 3'-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction. The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions. Micrococcus luteus UV endonuclease also nicks the C(3')-O-P bond 3' to AP sites by a beta-elimination reaction. No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation.     

Localization of the phosphoester bond hydrolyzed by the major apurinic/apyrmidinic endodeoxyribonuclease from rat-liver chromatin

The major apurinic/apyrimidinic (AP) endodeoxyribonuclease from rat liver chromatin, an enzyme specific for AP sites in DNA, cleaves the phosphodiester bridge which is the immediate neighbour of the AP site on its 5' side leaving 3'-hydroxyl and 5'-phosphate ends. In contrast with Escherichia coli endonuclease VI, this chromatin enzyme is inactive on reduced AP sites.     

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.     

Some characteristics of apurinic sites in single- and double-stranded biologically active phi X174 DNA

With biologically active DNA of the bacteriophage phi X174, both single and double-stranded, some physico-chemical and biological parameters of the depurination reaction are studied. It is shown that in single-stranded DNA each apurinic site is lethal, while in double-stranded RFI-DNA only about 5% of these sites are lethal. Furthermore it is concluded that the apurinic sites are formed at different rates in single- and double-stranded DNA and also the conversion into breaks of the apurinic sites is different for both forms of DNA.     

A single apurinic site can elicit BAL 31 nuclease-catalyzed cleavage in duplex DNA

The extracellular nuclease from Alteromonas espejiana BAL 31 is a highly sensitive endonucleolytic probe for lesions that distort the helical structure of duplex DNA. The nuclease can be isolated as two distinct molecular species, the 'fast' (F) and 'slow' (S) species, which have different kinetic properties. When nonsupercoiled, covalently closed circular phage PM2 DNA containing apurinic sites introduced by heating at acid pH was incubated with individual fractions from a chromatographic column which separated the two nuclease species, cleavage of the DNA was observed which was greatly in excess of control levels using nonmodified DNA. The initial rates of such cleavage clearly paralleled the peaks of both absorbance and nuclease activity against single-stranded and linear duplex substrates. When samples of apurinic DNA were incubated with pooled fractions from the same column representing pure F and S nucleases, respectively, the rate and extent of the cleavage observed was dependent upon the average number of apurinic sites per molecule. Cleavage was readily detectable in samples containing an average of 1.1 apurinic sites per molecule with both species of the enzyme. These results indicate that either species of the BAL 31 nuclease can recognize and cleave in response to a single apurinic site in duplex DNA. The F nuclease appears to be approx. 2.5-times as efficient in cleaving DNA containing apurinic lesions as the S enzyme in extended incubations.     

Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: design of a 32P-postlabeling assay for apurinic sites in DNA

We have examined the capacity of bacteriophage T4 polynucleotide kinase (EC 2.7.1.78) to phosphorylate the partially depurinated products of d-ApA, namely, d-SpA and d-ApS (where S represents an apurinic deoxyribose group). It was observed that the enzyme acted only on the latter isomer. Since molecules of this type (d-NpS) are the sole apurinic site containing products resulting from the combined digestion of lightly depurinated DNA by snake venom phosphodiesterase and calf alkaline phosphatase [Weinfeld, M., Liuzzi, M., & Paterson, M. C. (1989) Nucleic Acids Res. 17, 3735-3745], we were able to devise a postlabeling assay for these biologically important DNA lesions. The method offers several advantages, including (a) elimination of the need for prelabeled DNA, (b) high (femtomole range) sensitivity, and (c) nearest-neighbor analysis of bases 5' to apurinic/apyrimidinic sites. Using this assay, we obtained a value for the rate of depurination of form I pRSVneo plasmid DNA, incubated at pH 5.2 at 70 degrees C, of approximately 3.3 apurinic sites per plasmid molecule per hour. This value compares favorably with previously published data of others, acquired by alternative approaches. The rate of depurination of poly(dA), treated in a similar fashion, was found to be approximately 1 base per 10(3) nucleotides per hour.     

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.