The exoA gene of Streptococcus pneumoniae and its product, a DNA exonuclease with apurinic endonuclease activity.

The gene encoding the major DNA exonuclease of Streptococcus pneumoniae, exoA, was cloned in a streptococcal host vector system. Its location was determined by subcloning and by insertion mutations. Transfer of a DNA segment containing the gene to an Escherichia coli expression vector showed that exoA was the structural gene for the enzyme and that it was adjacent to its promoter. DNA sequence determination indicated that the gene encoded a protein, ExoA, of molecular weight 31,263. Under hyperexpression conditions, the ExoA protein constituted 10% of total cellular protein. In addition to previously demonstrated 3' to 5' exonuclease and 3'-phosphatase activities, ExoA was shown to make single-strand breaks at apurinic sites in DNA. Its enzymatic activities are thus similar to those of exonuclease III of E. coli and other gram-negative bacteria. The nucleotide sequence of exoA revealed it to be homologous to xth of E. coli, with 26% identity of amino acid residues in the predicted proteins. So far, no null chromosomal mutants of exoA have been obtained, and the biological function of ExoA remains unknown.     

Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst.

The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.     

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.     

Reassessment of the in vivo functions of DNA polymerase I and RNase H in bacterial cell growth

A major factor in removing RNA primers during the processing of Okazaki fragments is DNA polymerase I (Pol I). Pol I is thought to remove the RNA primers and to fill the resulting gaps simultaneously. RNase H, encoded by rnh genes, is another factor in removing the RNA primers, and there is disagreement with respect to the essentiality of both the polA and rnh genes. In a previous study, we looked for the synthetic lethality of paralogs in Bacillus subtilis and detected several essential doublet paralogs, including the polA ypcP pair. YpcP consists of only the 5'-3' exonuclease domain. In the current study, we first confirmed that the polA genes of both Escherichia coli and B. subtilis could be completely deleted. We found that the 5'-3' exonuclease activity encoded by either polA or ypcP xni was required for the growth of B. subtilis and E. coli. Also, the 5'-3' exonuclease activity of Pol I was indispensable in the cyanobacterium Synechococcus elongatus. These results suggest that a 5'-3' exonuclease activity is essential in these organisms. Our success in constructing a B. subtilis strain that lacked all RNase H genes indicates that the enzymatic activity is dispensable, at least in the wild type. Increasing the 5'-3' exonuclease activity partially compensated for a defective phenotype of an RNase H-deficient mutant, suggesting cooperative functions for the two enzyme systems. Our search for the distribution of the 5'-3' exonuclease domain among 250 bacterial genomes resulted in the finding that all eubacteria, but not archaea, possess this domain.     

Role of the Nfo (YqfS) and ExoA apurinic/apyrimidinic endonucleases in protecting Bacillus subtilis spores from DNA damage

The Bacillus subtilis enzymes ExoA and Nfo (originally termed YqfS) are endonucleases that can repair apurinic/apyrimidinic (AP) sites and strand breaks in DNA. We have analyzed how the lack of ExoA and Nfo affects the resistance of growing cells and dormant spores of B. subtilis to a variety of treatments, some of which generate AP sites and DNA strand breaks. The lack of ExoA and Nfo sensitized spores (termed alpha-beta-) lacking the majority of their DNA-protective alpha/beta-type small, acid-soluble spore proteins (SASP) to wet heat. However, the lack of these enzymes had no effect on the wet-heat resistance of spores that retained alpha/beta-type SASP. The lack of either ExoA or Nfo sensitized wild-type spores to dry heat, but loss of both proteins was necessary to sensitize alpha-beta- spores to dry heat. The lack of ExoA and Nfo also sensitized alpha-beta-, but not wild-type, spores to desiccation. In contrast, loss of ExoA and Nfo did not sensitize growing cells or wild-type or alpha-beta- spores to hydrogen peroxide or t-butylhydroperoxide. Loss of ExoA and Nfo also did not increase the spontaneous mutation frequency of growing cells. exoA expression took place not only in growing cells, but also in the forespore compartment of the sporulating cell. These results, together with those from previous work, suggest that ExoA and Nfo are additional factors that protect B. subtilis spores from DNA damage accumulated during spore dormancy.     

YqfS from Bacillus subtilis is a spore protein and a new functional member of the type IV apurinic/apyrimidinic-endonuclease family

The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His6-YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His6-YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3'-phosphatases. However, the purified protein lacked both 5'-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3' blocking groups of DNA generated during both spore dormancy and germination.     

Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants.

Apurinic/apyrimidinic (AP) sites in cellular DNA are considered to be both cytotoxic and mutagenic, and can arise spontaneously or following exposure to DNA damaging agents. We have isolated cDNA clones which encode an endonuclease, designated HAP1 (human AP endonuclease 1), that catalyses the initial step in AP site repair in human cells. The predicted HAP1 protein has an Mr of 35,500 and shows striking sequence similarity (93% identity) to BAP 1, a bovine AP endonuclease enzyme. Significant sequence homology to two bacterial DNA repair enzymes, E. coli exonuclease III and S. pneumoniae ExoA proteins, and to Drosophila Rrp1 protein is also apparent. We have expressed the HAP1 cDNA in E. coli mutants lacking exonuclease III (xth), endonuclease IV (nfo), or both AP endonucleases. The HAP1 protein can substitute for exonuclease III, but not for endonuclease IV, in respect of some, but not all, DNA repair and mutagenesis functions. Moreover, a dut xth (ts) double mutant, which is nonviable at 42 degrees C due to an accumulation of unrepaired AP sites following excision of uracil from DNA, was rescued by expression of the HAP1 cDNA. These results indicate that AP endonucleases show remarkable conservation of both primary sequence and function. We would predict that the HAP1 protein is important in human cells for protection against the toxic and mutagenic effects of DNA damaging agents.     

A versatile endonuclease IV from Thermus thermophilus has uracil-excising and 3'-5' exonuclease activity

Apurinic/apyrimidinic (AP) sites arise in DNA through the spontaneous loss of bases or through the release of damaged bases from DNA by DNA glycosylases. AP sites in DNA can be catalyzed by AP endonucleases such as exonuclease III and endonuclease IV, generating a 3'-hydroxyl group and a 5'-terminal sugar phosphate. Here, we have identified and characterized a novel endonuclease IV from a hyperthermophilic bacterium Thermus thermophilus designated as TthNfo. TthNfo efficiently removed AP site from double-stranded oligonucleotide substrate. No significant difference was observed in the rate of reaction of four bases opposite AP site with TthNfo. In addition, TthNfo possesses a 3'-5' exonuclease activity similar to that of Escherichia coli exonuclease III. Surprisingly, we found that TthNfo also catalyzes the excision of uracil from DNA. In comparison with other endonuclease IV proteins, the removal of uracil residue was unique to TthNfo. Based on these observations and the absence of exonuclease III in T. thermophilus, we suggest that versatile enzyme activities of TthNfo play an important role in counteracting DNA base damage in vivo.     

Exonuclease X of Escherichia coli. A novel 3'-5' DNase and Dnaq superfamily member involved in DNA repair.

DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacterium Escherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3'-5' exonuclease proofreading subunit (DnaQ) of E. coli DNA polymerase III. We have engineered and purified a (His)(6)-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3'-5' polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation.     

Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney.

Escherichia coli endonuclease IV hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free deoxyribose. It also hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free 2',3'-unsaturated sugar produced by nicking 3' to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3'-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3')-O-P bond 5' to the AP site is hydrolysed, but in a second step the 5'-terminal base-free deoxyribose 5'-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3')-O-P bond 3' to a 5'-terminal AP site contrasts with the relative stability of the same bond 3' to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5'-terminal AP site means that, after nicking 5' to an AP site with an AP endonuclease, in principle no 5'----3' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'----3' exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5'-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3'-phosphoglycollatase and also a 3'-phosphatase. The 3'-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.     

The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates

The DNA polymerase activity of the near homogeneous, multisubunit DNA polymerase-primase from Drosophila melanogaster embryos has been compared to Escherichia coli DNA polymerase III core, DNA polymerase III, and DNA polymerase III holoenzyme. The rate of deoxynucleotide incorporation by the Drosophila polymerase on singly primed phi X174 DNA is similar to that observed with equivalent levels of DNA polymerase III holoenzyme in the absence of E. coli single-stranded DNA binding protein. However, analysis of the DNA products indicates that the Drosophila polymerase is less processive than DNA polymerase III holoenzyme, and closely resembles DNA polymerase III. The Drosophila polymerase-primase contains neither 3'-5' exonuclease nor RNase H-like activities, and catalyzes no significant pyrophosphate exchange. There is a low level of DNA-dependent ATPase activity which can be eliminated by a second glycerol gradient sedimentation (Kaguni, L.S., Rossignol, J.-M., Conaway, R.C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2221-2225). Although lacking a 3'-5' exonuclease, the replication fidelity of the D. melanogaster polymerase is similar to that of E. coli DNA polymerase III holoenzyme which possesses such an activity.     

Exonuclease III from Bacillus amyloliquefaciens

Bacillus amyloliquefaciens cells were found to contain an exonuclease which catalyzes the sequential hydrolysis of mononucleotides from the 3'-termini of duplex DNA. The enzyme was purified to homogeneity and its molecular weight (as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) is 29,000. The exonuclease possesses an additional catalytic activity, i.e., 3'-5' exonuclease specific for the RNA strand in an RNA--DNA hybrid duplex (RNase H activity). In terms of physical and catalytic properties the exonuclease of B. amyloliquefaciens is similar to exonucleases III from E. coli and Haemophilus influenzae and can thus be related to the same class of hydrolases, i.e., 3.1.11.2. However, in comparison with exo III from E. coli, the enzyme from B. amyloliquefaciens exhibits a more strict specificity for the structure of the substrate 3'-end.     

cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III.

cDNA encoding the human homologue of mouse APEX nuclease was isolated from a human bone-marrow cDNA library by screening with cDNA for mouse APEX nuclease. The mouse enzyme has been shown to possess four enzymatic activities, i.e., apurinic/apyrimidinic endonuclease, 3'-5' exonuclease, DNA 3'-phosphatase and DNA 3' repair diesterase activities. The cDNA for human APEX nuclease was 1420 nucleotides long, consisting of a 5' terminal untranslated region of 205 nucleotide long, a coding region of 954 nucleotide long encoding 318 amino acid residues, a 3' terminal untranslated region of 261 nucleotide long, and a poly(A) tail. Determination of the N-terminal amino acid sequence of APEX nuclease purified from HeLa cells showed that the mature enzyme lacks the N-terminal methionine. The amino acid sequence of human APEX nuclease has 94% sequence identity with that of mouse APEX nuclease, and shows significant homologies to those of Escherichia coli exonuclease III and Streptococcus pneumoniae ExoA protein. The coding sequence of human APEX nuclease was cloned into the pUC18 SmaI site in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain and BW9109 (delta xth) strain cells of E. coli. The transformed cells expressed a 36.4 kDa polypeptide (the 317 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide derived from the part of pUC18 sequence), and were less sensitive to methylmethanesulfonate and tert-butyl-hydroperoxide than the parent cells. The N-terminal regions of the constructed protein and APEX nuclease were cleaved frequently during the extraction and purification processes of protein to produce the 31, 33 and 35 kDa C-terminal fragments showing priming activities for DNA polymerase on acid-depurinated DNA and bleomycin-damaged DNA. Formation of such enzymatically active fragments of APEX nuclease may be a cause of heterogeneity of purified preparations of mammalian AP endonucleases. Based on analyses of the deduced amino acid sequence and the active fragments of APEX nuclease, it is suggested that the enzyme is organized into two domains, a 6 kDa N-terminal domain having nuclear location signals and 29 kDa C-terminal, catalytic domain.     

Recognition of oxidized abasic sites by repair endonucleases.

The recognition of 'regular' and 'oxidized' sites of base loss (AP sites) in DNA by various AP endonucleases was compared. Model substrates with regular AP sites (resulting from mere hydrolysis of the glycosylic bond) were produced by damaging bacteriophage PM2 DNA by exposure to low pH; those with AP sites oxidized at the C-4'- and C-1'-position of the sugar moiety by exposure to Fe(III)-bleomycin in the presence of H2O2 and to Cu(II)-phenanthroline in the presence of H2O2 and ethanol, respectively. The results confirmed that AP sites-together with single-strand breaks-are indeed the predominant type of DNA modification in all three cases. For the recognition of 4'-oxidized AP sites, a 400-fold higher concentration of Escherichia coli exonuclease III and between 5-fold and 50-fold higher concentrations of bacteriophage T4 endonuclease V, E. coli endonuclease III and E. coli FPG protein were required than for the recognition of regular AP sites. In contrast, the recognition of 4'-oxidized AP sites by E. coli endonuclease IV was effected by 4-fold lower concentrations than needed for regular AP sites. 1'-oxidized AP sites (generated by activated Cu(II)-phenanthroline) were recognized by endonuclease IV and exonuclease III only slightly (3-fold and 13-fold, respectively) less efficiently than regular AP sites. In contrast, there was virtually no recognition of 1'-oxidized AP sites by the enzymes which cleave at the 3' side of AP sites (T4 endonuclease V, endonuclease III and FPG protein). The described differences were exploited for the analysis of the DNA damage induced by hydroxyl radicals, generated by ionizing radiation or Fe(III)-nitrilotriacetate in the presence of H2O2. The results indicate that both regular and 1'-oxidized AP sites represent only minor fractions of the AP sites induced by hydroxyl radicals.     

Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.     

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.     

Isolation of cDNA clones encoding an enzyme from bovine cells that repairs oxidative DNA damage in vitro: homology with bacterial repair enzymes.

Ionizing radiation and radiomimetic compounds, such as hydrogen peroxide and bleomycin, generate DNA strand breaks with fragmented deoxyribose 3' termini via the formation of oxygen-derived free radicals. These fragmented sugars require removal by enzymes with 3' phosphodiesterase activity before DNA synthesis can proceed. An enzyme that reactivates bleomycin-damaged DNA to a substrate for Klenow polymerase has been purified from calf thymus. The enzyme, which has a Mr of 38,000 on SDS-PAGE, also reactivates hydrogen peroxide-damaged DNA and has an associated apurinic/apyrimidinic (AP) endonuclease activity. The N-terminal amino acid sequence of the purified protein matches that reported previously for a calf thymus enzyme purified on the basis of AP endonuclease activity. Degenerate oligonucleotide primers based on this sequence were used in the polymerase chain reaction to generate from a bovine cDNA library a fragment specific for the 5' end of the coding sequence. Using this cDNA fragment as a probe, several clones containing 1.35 kb cDNA inserts were isolated and the complete nucleotide sequence of one of these determined. This revealed an 0.95 kb open reading frame which would encode a polypeptide of Mr 35,500 and with a N-terminal sequence matching that determined experimentally. The predicted amino acid sequence shows strong homology with the sequences of two bacterial enzymes that repair oxidative DNA damage, ExoA protein of S. pneumoniae and exonuclease III of E. coli.     

cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III

We purified a mouse DNA repair enzyme having apurinic/apyrimidinic endonuclease, DNA 3'-phosphatase, 3'-5'-exonuclease and DNA 3' repair diesterase activities, and designated the enzyme as APEX nuclease. A cDNA clone for the enzyme was isolated from a mouse spleen cDNA library using probes of degenerate oligonucleotides deduced from the N-terminal amino acid sequence of the enzyme. The complete nucleotide sequence of the cDNA (1.3 kilobases) was determined. Northern hybridization using this cDNA showed that the size of its mRNA is about 1.5 kilobases. The complete amino acid sequence for the enzyme predicted from the nucleotide sequence of the cDNA (APEX nuclease cDNA) indicates that the enzyme consists of 316 amino acids with a calculated molecular weight of 35,400. The predicted sequence contains the partial amino acid sequences determined by a protein sequencer from the purified enzyme. The coding sequence of APEX nuclease was cloned into pUC18 SmaI and HindIII sites in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain cells of Escherichia coli. The transformed cells expressed a 36.4-kDa polypeptide (the 316 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide of beta-galactosidase) and were less sensitive to methyl methanesulfonate than the parent cells. The fusion product showed priming activity for DNA polymerase on bleomycin-damaged DNA and acid-depurinated DNA. The deduced amino acid sequence of mouse APEX nuclease exhibits a significant homology to those of exonuclease III of E. coli and ExoA protein of Streptococcus pneumoniae and an intensive homology with that of bovine AP endonuclease 1.     

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3''-5'' exonuclease active site.

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.     

Gaps in DNA induced by neocarzinostatin bear 3'- and 5'-phosphoryl termini.

Neocarzionstatin (NCS)-induced strand breakage of DNA generates nonfunctional binding sites for the E. coli DNA polymerase I. Treatment of the NCS-nicked DNA with alkaline phosphatase at 65 degrees C prior to the polymerase reaction results in 60-100-fold stimulation of dTMP incorporation whereas in a control not treated with the drug there is only a 2-fold increase. Sites of strand scission on the NCS-treated DNA bear phosphate at the 3' termini. This conclusion is supported by the kinetics of release of inorganic phosphate from NCS-cut DNA by exonuclease III. Since our earlier work has shown that virtually all the 5' ends of the nicks caused by NCS bear phosphomonoester groupings, the 3'- and 5'- phosphoryl termini could be quantitated using alkaline phosphatase and exonuclease III. Over a wide range of drug levels the amount of inorganic phosphate released by alkaline phosphatase is approximately twice as much as that removed by exonuclease III, indicating the presence of equal amounts of 3'- and 5'- phosphoryl termini. This, taken together with other previously demonstrated effects of NCS on DNA, such as the introduction of nicks not sealable by polynucleotide ligase, the release of thymine, and the formation of a malonaldehyde type compound, suggests that NCS-induced strand breakage involves base release accompanied by opening of the sugar ring with destruction of one or more nucleosides and results in a gap bounded by 3'- and 5'- phosphoryl termini.