Yeast DNA 3'-repair diesterase is the major cellular apurinic/apyrimidinic endonuclease: substrate specificity and kinetics

DNA strand breaks with damaged 3' termini are potentially toxic lesions caused by free radicals. The purified yeast diesterase that removes small nucleotide fragments from such 3' termini in oxidized DNA has been further characterized with respect to its substrate specificity. In addition to the 3'-phosphoglycolaldehyde esters used to monitor the activity during purification, the enzyme efficiently hydrolyzed a variety of other 3'-esters in DNA. These included 3'-phosphates, 3'-(2,3-didehydro-2,3-dideoxyribose phosphates), and the 3'-blocking damages formed in vivo in Escherichia coli by H2O2 or in vitro by DNA treatment with bleomycin. This same transition metal-dependent enzyme also constitutes the major yeast endonuclease for apurinic/apyrimidinic sites in DNA, hydrolyzing these damages to yield normal 3'-hydroxyl nucleotides and 5'-phosphoryl base-free sugar termini (a Type II apurinic/apyrimidinic endonuclease). Yeast 3'-phosphoglycolaldehyde diesterase therefore appears to be involved in two distinct pathways of DNA repair: initiation of the repair of oxidative strand breaks in DNA and the restoration of sites of base loss caused by many types of DNA-damaging agents.     

A mutant endonuclease IV of Escherichia coli loses the ability to repair lethal DNA damage induced by hydrogen peroxide but not that induced by methyl methanesulfonate.

A mutant allele of the Escherichia coli nfo gene encoding endonuclease IV, nfo-186, was cloned into plasmid pUC18. When introduced into an E. coli xthA nfo mutant, the gene product of nfo-186 complemented the hypersensitivity of the mutant to methyl methanesulfonate (MMS) but not to hydrogen peroxide (H2O2) and bleomycin. These results suggest that the mutant endonuclease IV has normal activity for repairing DNA damages induced by MMS but not those induced by H2O2 and bleomycin. A missense mutation in the cloned nfo-186 gene, in which the wild-type glycine 149 was replaced by aspartic acid, was detected by DNA sequencing. The wild-type and mutant endonuclease IV were purified to near homogeneity, and their apurinic (AP) endonuclease and 3'-phosphatase activities were determined. No difference was observed in the AP endonuclease activities of the wild-type and mutant proteins. However, 3'-phosphatase activity was dramatically reduced in the mutant protein. From these results, it is concluded that the endonuclease IV186 protein is specifically deficient in the ability to remove 3'-terminus-blocking damage, which is required for DNA repair synthesis, and it is possible that the lethal DNA damage by H2O2 is 3'-blocking damage and not AP-site damage.     

Metalloenzymes in DNA repair. Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1.

Escherichia coli endonuclease IV and its Saccharomyces cerevisiae homologue Apn1, two DNA repair enzymes for free radical damages, were previously shown to be inactivated by metal-chelating agents. In the present study, atomic absorption spectrometry of endonuclease IV revealed the presence of 2.4 zinc and 0.7 manganese atoms, whereas Apn1 contained 3.3 zinc atoms and no significant manganese. EDTA-inactivated endonuclease IV retained 0.7 zinc atom but little detectable manganese. ZnCl2 reactivated 1,10-phenanthroline-treated Apn1, but was ineffective with endonuclease IV treated with either 1,10-phenanthroline or EDTA. In contrast, enzymatic activity was restored to both enzymes after EDTA treatment by incubation with CoCl2 and to a lesser extent by MnCl2. Endonuclease IV, reactivated with CoCl2 or MnCl2, regained all of the activities characteristic of the native enzyme. MnCl2 was as effective as CoCl2 at restoring activity to the 1,10-phenanthroline-treated enzymes. The results indicate that intrinsic metals play critical roles in both endonuclease IV and Apn1 and that manganese may perform a special function in endonuclease IV. Possible mechanistic roles for the metals in these DNA repair enzymes are discussed.     

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.     

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.     

Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants.

Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.     

In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo.

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3'-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo-) and endonuclease IV-overproducing (p-nfo; approximately 10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo- strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo- cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo- strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was > or = 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.     

Endonuclease IV (nfo) mutant of Escherichia coli.

A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested.     

Endonuclease IV homolog from Dictyostelium discoideum: sequencing and functional expression in AP endonuclease-deficient Escherichia coli

We sequenced a gene encoding AP endonuclease DdAPN in Dictyostelium discoideum. The sequence predicts a protein of 542 amino acids, showing high homology to Escherichia coli Endonuclease IV (Endo IV). There is 45% identity to Endo IV using the C-terminal 282 amino acids of the Dictyostelium protein. The DdAPN conserves nine residues for the metal-binding identified in Endo IV. The truncated DdAPN protein containing these sites partially complemented E. coli RPC501 (xth(-), nfo(-)).     

Repair of DNA lesions induced by hydrogen peroxide in the presence of iron chelators in Escherichia coli: participation of endonuclease IV and Fpg

In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product. Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions. Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.     

Multiple pathways for repair of oxidative DNA damages caused by X rays and hydrogen peroxide in Escherichia coli.

The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.     

Development of T7 phage and T7 phage containing apurinic sites in an exonuclease III, endonuclease IV double mutant of Escherichia coli.

The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo). In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis. These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair. In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.     

Purification and characterization of Thermotoga maritima endonuclease IV, a thermostable apurinic/apyrimidinic endonuclease and 3'-repair diesterase

An endonuclease IV homolog was identified as the product of a conceptual open reading frame in the genome of the hyperthermophilic bacterium Thermotoga maritima. The T. maritima endonuclease IV gene encodes a 287-amino-acid protein with 32% sequence identity to Escherichia coli endonuclease IV. The gene was cloned, and the expressed protein was purified and shown to have enzymatic activities that are characteristic of the endonuclease IV family of DNA repair enzymes, including apurinic/apyrimidinic endonuclease activity and repair activities on 3'-phosphates, 3'-phosphoglycolates, and 3'-trans-4-hydroxy-2-pentenal-5-phosphates. The T. maritima enzyme exhibits enzyme activity at both low and high temperatures. Circular dichroism spectroscopy indicates that T. maritima endonuclease IV has secondary structure similar to that of E. coli endonuclease IV and that the T. maritima endonuclease IV structure is more stable than E. coli endonuclease IV by almost 20 degrees C, beginning to rapidly denature only at temperatures approaching 90 degrees C. The presence of this enzyme, which is part of the DNA base excision repair pathway, suggests that thermophiles use a mechanism similar to that used by mesophiles to deal with the large number of abasic sites that arise in their chromosomes due to the increased rates of DNA damage at elevated temperatures.     

Chlamydia pneumoniae AP endonuclease IV could cleave AP sites of double- and single-stranded DNA

Endonuclease IV gene, the only putative AP endonuclease of C. pneumoniae genome, was cloned into pET28a. Recombinant C. pneumoniae endonuclease I V (CpEndoIV) was expressed in E. coli and purified to homogeneity. CpEndoIV has endonuclease activity against apurinic/apyrimidinic sites (AP sites) of double-stranded (ds) oligonucleotides. AP endonuclease activity of CpEndoIV was promoted by divalent metal ions Mg2+ and Zn2+, and inhibited by EDTA. The natural (A, T, C and G) and modified (U, I and 8-oxo-G (GO)) bases opposite AP site had little effect on the cleavage efficiency of AP site of ds oligonucleotides by CpEndoIV. However, the CpEndoIV-dependent cleavage of AP site opposite modified base GO was strongly inhibited by Chlamydia DNA glycosylase MutY. Interestingly, the AP site in single-stranded (ss) oligonucleotides was also the effective substrate of CpEndoIV. Similar to E. coli endonuclease IV, AP endonuclease activity of CpEndoIV was also heat-stable to some extent, with a half time of 5 min at 60 degrees C.     

Characterization of an endonuclease IV 3'-5' exonuclease activity

Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3' DNA blocking groups. By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV. Here we report that endonuclease IV does possess an intrinsic 3'-5' exonuclease activity. The activity was detected in purified preparations of the endonuclease IV protein from E. coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions. Induction of either endonuclease IV in an E. coli overexpression system resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the endonuclease IV AP endonuclease activity. Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3' recessed ends were preferred substrates for the 3'-5' exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo.     

Further purification and characterization of T4 endonuclease V.

T4 endonuclease V, which is involved in repair of ultraviolet-damaged DNA, has been purified 3600 fold from T4D-infected Escherichia coli. The enzyme shows optimal activity at pH 7.2 and does not require added divalent ions. Endonuclease V attacks both native and heat-denatured DNA provided that the DNA has been irradiated, and the enzyme activity is dependent on the dose of ultraviolet irradiation. The rate and the extent of the reaction are greater with irradiated native DNA although the Km values for the two types of DNA are the same (2.25 - 10(-5) M). The enzyme is readily inactivated by heat and is sensitive to p-chloromercuribenzoate. Endonuclease V-treated irradiated DNA is degraded by spleen phosphodiesterase only when the DNA has been treated with alkaline phosphatase, suggesting that the enzyme produces 5'-phosphoryl termini.     

Bacteriophage T4 Endonucleases II and Iv, Oppositely Affected by Dcmp Hydroxymethylase Activity, Have Different Roles in the Degradation and in the RNA Polymerase-Dependent Replication of T4 Cytosine-Containing DNA

Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt). This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.-Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA. The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions. Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA. Possible mechanisms for this inhibition are discussed.-The E. coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template. In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template. Cyt-DNA synthesis dependent on the unmodified (33-55-) RNA polymerase was less sensitive to endonuclease IV action.     

Drosophila Rrp1 complements E. coli xth nfo mutants: protection against both oxidative and alkylation-induced DNA damage.

Drosophila Rrp1 protein has four tightly associated enzymatic activities: DNA strand transfer, ssDNA renaturation, dsDNA 3'-exonuclease and apurinic/apyrimidinic (AP) endonuclease. The carboxy-terminal region of Rrp1 is homologous to Escherichia coli exonuclease III and several eukaryotic AP endonucleases. All members of this protein family cleave abasic sites. Rrp1 protein was expressed under the control of the E. coli RNA polymerase tac promoter (pRrp1-tac) in two repair deficient E. coli strains (BW528 and LG101) lacking both exonuclease III (xth) and endonuclease IV (nfo). Rrp1 confers resistance to killing by oxidative, antitumor and alkylating agents that damage DNA (hydrogen peroxide, t-butylhydroperoxide, bleomycin, methyl methanesulfonate, and mitomycin C). Complementation of the repair deficiency by Rrp1 provides up to a two log increase in survival and requires the C-terminal nuclease region of Rrp1, but not its N-terminal region. The AP endonuclease activity in extracts from the repair deficient strain LG101 is increased up to 12-fold when the strain contains pRrp1-tac. These results indicate that pRrp1-tac directs the synthesis of active enzyme, and that the nuclease activities of Rrp1 are likely to be the cause of the increased resistance to DNA damage of the mutant cells.     

Yeast DNA diesterase for 3'-fragments of deoxyribose: purification and physical properties of a repair enzyme for oxidative DNA damage

The DNA strand breaks resulting from exposure to the free radicals generated by ionizing radiation or oxidizing agents are refractory to DNA repair synthesis because of deoxyribose fragments that block their 3' termini. The restoration of normal 3'-OH nucleotide primers is the essential first step in the excision repair of these radical-induced strand breaks. We have used a synthetic DNA substrate containing 3'-phosphoglycolaldehyde esters to identify and purify to physical homogeneity the major yeast diesterase that removes such nucleotide fragments. Yeast 3'-phosphoglycolaldehyde diesterase had Mr = 40,500 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular weight estimate from gel filtration indicated that the active species is a nearly globular monomer. Purification of the enzyme removed a tightly bound metal, but the activity of the purified enzyme could be restored by the addition of Co2+, Mn2+, Ni2+, or Zn2+, with Co2+ the most effective cofactor. Even 3 microM Co2+ stimulated near-maximal activity, and this metal also conferred significant thermal stability on the purified protein. This is a novel enzyme, whose N-terminal amino acid sequence does not show any significant similarity to published sequences, and which is not the product of any gene in the RAD52 epistasis group.     

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.