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.     

Excision of apurinic and/or apyrimidinic sites from DNA by nucleolytical enzymes from rat brain

Apurinic and/or apyrimidinic (AP) sites were excised from PM2 phage DNA by two enzymes: an AP endodeoxyribonuclease isolated from rat neocortex chromatin and a rat brain exodeoxyribonuclease, DNase B III. The resulting gap was filled with DNA polymerase beta prepared from rat liver and finally ligated by Escherichia coli DNA ligase.     

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.     

Molecular mechanisms of mutagenesis caused by apurinic/apyrimidinic DNA sites

The subject of this review are molecular mechanisms and specificity of mutagenesis induced by apurinic/apyrimidinic (AP) sites representing a characteristic group of so called non-coding DNA lesions. The data available suggest that efficiency and specificity of AP sites-induced mutations depend, primarily, on genome structural organization. This is manifested in existence of DNA sequences highly prone to depurination/depyrimidination as well as in the ability of specific DNA regions to adopt potentially mutagenic conformations. The latter leads to mutations as consequence of AP sites' repair. Secondly, the AP sites-induced mutagenesis depends on functional state of genome, on the ability of replicative/repair cell apparatus to carry out some specific forms of mutagenic DNA repair, in particular, to bypass non-coding DNA lesions under conditions of SOS repair.     

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.     

A new endonuclease from Escherichia coli acting at apurinic sites in DNA.

A new DNA endonuclease has been purified 3000-fold from Escherichia coli. The enzyme specifically catalyzes the formation of single strand breaks at apurinic and apyrimidinic sites in DNA, but has no activity on intact or single-stranded DNA. Further, the enzyme shows little or no activity on heavily ultraviolet-irradiated DNA, but cleaves x-irradiated DNA, presumably at apurinic and apyrimidinic sites introduced by the radiation treatment. The enzyme, which is tentatively named endonuclease IV, has no detectable associated exonuclease or DNA N-glycosidase activity and does not seem to be identical with any previously known E. coli endonuclease. Endonuclease IV has no Mg2+ requirement, and is fully active in the presence of EDTA. Enzyme activity is stimulated by 0.2 to 0.3 M NaCl and is unusually salt-resistant. Further, the enzyme is fairly heat-stable, and is not inhibited by tRNA. The sidimentation coefficient, S(o)20,w, is 3.4 S. It seems that endonuclease IV is active in DNA repair.     

Delta-elimination in the repair of AP (apurinic/apyrimidinic) sites in DNA.

[5'-32P]pdT8d(-)dT7, containing an AP (apurinic/apyrimidinic) site in the ninth position, and [d(-)-1',2'-3H, 5'-32P]DNA, containing AP sites labelled with 3H in the 1' and 2' positions of the base-free deoxyribose [d(-)] and with 32P 5' to this deoxyribose, were used to investigate the yields of the beta-elimination and delta-elimination reactions catalysed by spermine, and also the yield of hydrolysis, by the 3'-phosphatase activity of T4 polynucleotide kinase, of the 3'-phosphate resulting from the beta delta-elimination. Phage-phi X174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: beta-elimination, delta-elimination, hydrolysis of 3'-phosphate, DNA polymerization and ligation. Spermine, in one experiment, and Escherichia coli formamidopyrimidine: DNA glycosylase, in another experiment, were used to catalyse the first and second steps (beta-elimination and delta-elimination). These repair pathways, involving a delta-elimination step, may be operational not only in E. coli repairing its DNA containing a formamido-pyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites.     

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.     

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.     

Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli

The genetic requirements for the excision repair of thymine glycols, urea residues, and apurinic (AP) sites were examined by measuring the survival in Escherichia coli mutants of phi X174 replicative form (RF) I transfecting DNA containing selectively introduced lesions. phi X RF I DNA containing thymine glycols was inactivated at a greater rate in mutants deficient in endonuclease III (nth) than in wild-type hosts, suggesting that endonuclease III is involved in the repair of thymine glycols in vivo. phi X RF I DNA containing thymine glycols was also inactivated at a greater rate in mutants that were deficient in both exonuclease III and endonuclease IV (xth nfo) than in wild-type hosts, suggesting that a class II AP endonuclease is required for the in vivo processing of thymine glycols. phi X duplex-transfecting DNA containing urea residues or AP sites was inactivated at a greater rate in xth nfo double mutants than in wild-type, but not single-mutant, hosts, suggesting that exonuclease III or endonuclease IV is required for the repair of these damages and that either activity can substitute for the other. These data are in agreement with the known in vitro substrate specificities of endonuclease III, exonuclease III, and endonuclease IV.     

DNA deoxyribophosphodiesterase of Escherichia coli is associated with exonuclease I.

DNA deoxyribophosphodiesterase (dRpase) of E. coli catalyzes the release of deoxyribose-phosphate moieties following the cleavage of DNA at an apurinic/apyrimidinic (AP) site by either an AP endonuclease or AP lyase. Exonuclease I is a single-strand specific DNA nuclease which affects the expression of recombination and repair pathways in E. coli. We show here that a major dRpase activity in E. coli is associated with the exonuclease I protein. Highly purified exonuclease I isolated from an over-producing stain contains high levels of dRpase activity; it catalyzes the release of deoxyribose-5-phosphate from an AP site incised with endonuclease IV of E. coli and the release of 4-hydroxy-2-pentenal-5-phosphate from an AP site incised by the AP lyase activity of endonuclease III of E. coli. A strain containing a deletion of the sbcB gene showed little dRpase activity; the activity could be restored by transformation of the strain with a plasmid containing the sbcB gene. The dRpase activity isolated from an overproducing stain was increased 70-fold as compared to a normal sbcB+ strain (AB3027). These results suggest that the dRpase activity may be important in pathways for both DNA repair and recombination.     

Tyrosyl-DNA phosphodiesterase 1 initiates repair of apurinic/apyrimidinic sites

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.     

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.     

The enzymology of apurinic/apyrimidinic endonucleases.

Studies on the enzymology of apurinic/apyrimidinic (AP) endonucleases from procaryotic and eucaryotic organisms are reviewed. Emphasis will be placed on the enzymes from Escherichia coli from which a considerable portion of our knowledge has been derived. Recent studies on similar enzymes from eucaryotes will be discussed as well. In addition, we will discuss the chemical and physical properties of AP sites and review studies on peptides and acridine derivatives which incise DNA at AP sites.     

Escherichia coli single-stranded DNA binding protein stimulates the DNA deoxyribophosphodiesterase activity of exonuclease I.

The E. coli single-stranded binding protein (SSB) has been demonstrated in vitro to be involved in a number of replicative, DNA renaturation, and protective functions. It was shown previously that SSB can interact with exonuclease I to stimulate the hydrolysis of single-stranded DNA. We demonstrate here that E. coli SSB can also enhance the DNA deoxyribophosphodiesterase (dRpase) activity of exonuclease I by stimulating the release of 2-deoxyribose-5-phosphate from a DNA substrate containing AP endonuclease-incised AP sites, and the release of 4-hydroxy-2-pentenal-5-phosphate from a DNA substrate containing AP lyase-incised AP sites. E. coli SSB and exonuclease I form a protein complex as demonstrated by Superose 12 gel filtration chromatography. These results suggest that SSB may have an important role in the DNA base excision repair pathway.     

Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin

Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.     

Purification and properties of two endonucleases specific for apurinic/apyrimidinic sites in DNA from Saccharomyces cerevisiae

Two distinct endonucleases from Saccharomyces cerevisiae, specific for apurinic/apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from unspecific and ultraviolet-specific endonucleases and exonucleases. The two enzymes are monomeric proteins of around 24 000 daltons. Both are sensitive to ionic strength and most active in the presence of 150 and 100 mM NaCl for AP-endonucleases A and B, respectively. They are not absolutely dependent on divalent cations, since they are insensitive to EDTA, although AP-endonuclease A is activated by Ca²⁺ or Mg²⁺ and AP-endonuclease B by Mg²⁺ only. ATP inhibits the enzymes. AP-endonuclease A reacts optimally between pH 6 and 8, and AP-endonuclease B at pH 8. AP-endonuclease A is more stable at 60°C (half-life of 17 min) than B (half-life of 4 min). AP-endonucleuase A is insensitive to N-ethylmaleimide or ρ-chloromercuribenzoate. AP-endonuclease B is also insensitive to N-ethylmaleimide, but ρ-chloromercuribenzoate inhibits its activity.     

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.