Structure and Biological Activity of Deoxyribonucleic Acid from Bacillus Bacteriophage φ105: Effects of Escherichia coli Exonucleases

The effects of Escherichia coli exonuclease I, exonuclease III, and deoxyribonucleic acid (DNA) polymerase on the biological activity of mature DNA from temperate Bacillus bacteriophage phi105 were investigated. Intact DNA loses infectivity rapidly upon exposure to exonuclease III. Although there is an overall decrease in marker rescue from exonuclease III-digested DNA, digestion preferentially affects markers at the end of the genetic map. This is taken to indicate a nonpermuted gene sequence in mature DNA. Incubation of mature DNA in the presence of exonuclease I or DNA polymerase has no effect on its biological activity. The possible structure of the ends of mature phi105 DNA is discussed. The rate of digestion of mature phi105 DNA by exonuclease III is only about 1/20 the rate of lambda DNA. Results of digestion of various DNA substrates by exonuclease III indicate that the enzyme distinguishes between different DNA terminal structures.     

Proofreading activity of DNA polymerase III responds like elongation activity to auxiliary subunits

A comparison of the 3'----5' proofreading properties between Escherichia coli DNA polymerase III holoenzyme and DNA polymerase III' was conducted. This study indicated that the influence of the holoenzyme auxiliary subunits on the proofreading exonuclease parallels their effect on the elongation reaction. At physiological ionic strengths the auxiliary subunits markedly stimulated the exonuclease rate in an ATP-dependent reaction, while the exonuclease rate of DNA polymerase III' was not affected by ATP. E. coli single-stranded DNA binding protein stimulated the 3'----5' exonuclease activity of holoenzyme and inhibited DNA polymerase III'. Similarly, the auxiliary subunits and ATP converted the proofreading activity to a highly processive exonuclease. Our observation, that the exonuclease activity of the DNA polymerase III holoenzyme responded to ATP, salt, and E. coli single-stranded DNA-binding protein like the elongation activity, is consistent with the polymerase and exonuclease subunits acting within the same complex in a coordinated reaction.     

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

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

Determination of inhibition sites during hydrolysis of polydeoxyribonucleotides by exonucleases III from Bacillus amyloliquefaciens and Escherichia coli

The influence of the primary structure of polydeoxyribonucleotides on the rate of hydrolysis with exonuclease III from Bacillus amyloliquefaciens and Escherichia coli was investigated. The substrates used were synthetic oligodeoxyribonucleotides and pBR 322 DNA fragments labeled with 32P at the 5'-termini of one of the chains. According to the data from polyacrylamide gel electrophoresis performed under denaturing conditions, the hydrolysis of these substrates by unsaturating concentrations of B. amyloliquefaciens and E. coli exonuclease III proceeds with several reproducible "stops". The decrease of the reaction rate was shown to take place just before the pyrimidine blocks in the digested DNA chain.     

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.     

Sequence specificity of exonuclease III from E. coli.

The influence of the nucleotide sequence on the digestion of deoxyribonuclease from E. coli, has been investigated. It was found that the rate at which mononucleotides are released varies in a sequence dependent fashion. C-residues are cleaved off rapidly and G-residues slowly while A and T are released at an intermediate rate. Quantitative analyses of digestion experiments with synthetic DNA fragments made it possible to determine rate constants for the cleavage of several dinucleotide bonds by exonuclease III. These values were found to differ by up to a factor of 3. Summation of the differences can lead to appreciable variation in the overall rate of digestion of a DNA strand. The nucleotide specificity of exonuclease III leads to a transient appearance of a series of discrete DNA fragments intermediate in digestion and a stable set of fragments in limit digests, i.e. at the point when all DNA has become single-stranded. This property of exonuclease III needs to be taken into account for the application of the enzyme in the analysis of nucleoprotein complexes.     

Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved 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.     

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.     

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.     

Processivity of DNA exonucleases.

A homopolymer system has been developed to examine the digestion strategies of DNA exonucleases. Escherichia coli exonuclease I and lambda-exonuclease, are processive enzymes. However, T7 exonuclease, spleen exonuclease, E. coli exonuclease III, the 3' leads to 5'-exonuclease of T4 DNA polymerase, and both the 3' leads to 5' and the 5' leads to 3' activity of E. coli DNA polymerase I dissociate frequently from the substrate during the course of digestion. Regions of duplex DNA are a dissociation signal for exonuclease I.     

Normal processing of AP sites in Apn1-deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III

Escherichia coli exonuclease III and endonuclease III are two distinct DNA-repair enzymes that can cleave apurinic/apyrimidinic (AP) sites by different mechanisms. While the AP endonuclease activity of exonuclease III generates a 3'-hydroxyl group at AP sites, the AP lyase activity of endonuclease III produces a 3'-α,β unsaturated aldehyde that prevents DNA-repair synthesis. Saccharomyces cerevisiae Apn1 is the major AP endonuclease/3'-diesterase that also produces a 3'-hydroxyl group at the AP site, but it is unrelated to either exonuclease III or endonuclease III. apn1 deletion mutants are unable to repair AP sites generated by the alkylating agent methyl methane sulphonate and display a spontaneous mutator phenotype. This work shows that either exonuclease III or endonuclease III can functionally replace yeast Apn1 in the repair of AP sites. Two conclusions can be derived from these findings. The first of these conclusions is that yeast cells can complete the repair of AP sites even though they are cleaved by AP lyase. This implies that AP lyase can contribute significantly to the repair of AP sites and that yeast cells have the ability to process the α,β unsaturated aldehyde produced by endonuclease III. The second of these conclusions is that unrepaired AP sites are strictly the cause of the high spontaneous mutation rate in the apn1 deletion mutant.     

Characterization of Bacillus subtilis ExoA protein: a multifunctional DNA-repair enzyme similar to the Escherichia coli exonuclease III.

To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E. coli exonuclease III.     

Repair of x-ray-induced deoxyribonucleic acid single-strand breaks in xth mutants of Escherichia coli.

An exonuclease III-deficient strain of Escherichia coli K-12, BW2001 (xthA11), was unable to perform rapid repair of X-ray-induced deoxyribonucleic acid single-strand breaks and appeared to have a defect in the priming of the 3'-termini necessary for initiation of repair synthesis at the breaks. This defect cannot be explained solely by the lack of exonuclease III activity, because other xth mutants tested, including a deletion mutant, repaired radiation-induced strand breaks at close to the normal rate.     

Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease III.

Photoalkylation, the ultraviolet irradiation of DNA with isopropanol and di-tert-butylperoxide, causes a variety of base alterations. These include 8-(2-hydroxy-2-propyl)guanines, 8-(2-hydroxy-2-propyl)adenines and thymine dimers. An E. coli endonuclease against photoalkylated DNA was assayed by conversion of superhelical PM2 phage DNA to the nicked form. Enzyme activities were compared between extracts of strain BW9109 (xth-), lacking exonuclease III activity, and strain BW434 (xth-,nth-), deficient in both exonuclease III and endonuclease III. The endonuclease level in the double mutant against substrate photoalkylated DNA was under 20% of the activity in the mutant lacking only exonuclease III. Irradiation of the DNA substrate in the absence of isopropanol did not affect the activity in either strain. Analysis by polyacrylamide gel electrophoresis identified the sites of DNA cleavage by purified E. coli endonuclease III as cytosines, both in DNA irradiated at biologically significant wavelengths and in photoalkylated DNA. Neither 8-(2-hydroxy-2-propyl)purines, pyrimidine dimers, uracils nor 6-4'-(pyrimidin-2'-one)pyrimidines were substrates for the enzyme.     

Repair of oxidized abasic sites by exonuclease III, endonuclease IV, and endonuclease III

2-Deoxyribonolactone (L) and the C4'-oxidized abasic site (C4-AP) are produced by a variety of DNA-damaging agents. If not repaired, these lesions can be mutagenic. Exonuclease III and endonuclease IV are the major enzymes in E. coli responsible for 5'-incision of abasic sites (APs), the first steps in AP repair. Endonuclease III efficiently excises AP lesions via intermediate Schiff-base formation. Incision of L and C4-AP lesions by exonuclease III and endonuclease IV was determined under steady-state conditions using oligonucleotide duplexes containing the lesions at defined sites. An abasic lesion (AP) in an otherwise identical DNA sequence was incised by exonuclease III or endonuclease IV approximately 6-fold more efficiently than either of the oxidized abasic sites (L, C4-AP). Endonuclease IV incision efficiency of 2-deoxyribonolactone or C4-AP was independent of whether the lesion was opposite dA or dG. 2-Deoxyribonolactone is known to cross-link to endonuclease III (Hashimoto, M. (2001) J. Am. Chem. Soc. 123, 3161.). However, the C4-AP lesion is efficiently excised by endonuclease III. Oxidized abasic site repair by endonuclease IV and endonuclease III (C4-AP only) is approximately 100-fold less efficient than repair by exonuclease III. These results suggest that the first step of C4-AP and L oxidized abasic site repair will be the same as that of regular AP lesions in E. coli.     

Real-time direct observation of single-molecule DNA hydrolysis by exonuclease III

Single-molecule DNA digestion by exonuclease III, which has 3' to 5' exonuclease activity, was analyzed using a micro-channel with two-layer laminar flow. First, a DNA-bead complex was optically trapped in one layer in the absence of exonuclease III permitted the DNA to be stretched by the laminar flow. The exonuclease III reaction was initiated by moving the trapped DNA-bead complex to another layer of flow, which contained exonuclease III. As the reaction proceeded, the fluorescently-stained DNA was observed to shorten. The process was photographed; examination of the photographs showed that the DNA molecule shortened in a linear fashion with respect to the reaction time. The digestion rate obtained from the single-molecule experiment was compared to that measured from a bulk experiment and was found to be ca. 28 times higher than the bulk digestion rate.     

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.     

Mutations in Escherichia coli altering an apurinic endonuclease, endonuclease II, and exonuclease III and their effect on in vivo sensitivity to methylmethanesulfonate.

The levels of endonuclease II, an apurinic endonuclease, and exonuclease III in the parent strains (AB 1157) of Escherichia coli and in various mutants were determined by chromatography on DEAE-cellulose. AB 3027 and NH 5016 lacked endonuclease II and exonuclease III. BW 2001 lacked the apurinic endonuclease and exonuclease III while BW 2007, BW 9093, and BW 9059 lacked only exonuclease III. Deletion mutants BW 9101 and BW 9109 lacked all three enzymes. The latter mutants locate the genes for the two endonucleases in the region of exonuclease III (chith) of 38.2 min (White et al., 1976). All of the mutants which were sensitive to methylmethanesulfonate in vivo lacked exonuclease III, but not all mutants lacking exonuclease III were MMS sensitive. The deletion mutants and NH 5016 were the exceptions.     

Release of 5'-terminal deoxyribose-phosphate residues from incised abasic sites in DNA by the Escherichia coli RecJ protein.

Excision of deoxyribose-phosphate residues from enzymatically incised abasic sites in double-stranded DNA is required prior to gap-filling and ligation during DNA base excision-repair, and a candidate deoxyribophosphodiesterase (dRpase) activity has been identified in E. coli. This activity is shown here to be a function of the E. coli RecJ protein, previously described as a 5'-->3' single-strand specific DNA exonuclease involved in a recombination pathway and in mismatch repair. Highly purified preparations of dRpase contained 5'-->3' exonuclease activity for single-stranded DNA, and homogeneous RecJ protein purified from an overproducer strain had both 5'-->3' exonuclease and dRpase activity. Moreover, E. coli recJ strains were deficient in dRpase activity. The hydrolytic dRpase function of the RecJ protein requires Mg2+; in contrast, the activity of E. coli Fpg protein, that promotes the liberation of 5'-->3'Rp residues from DNA by beta-elimination, is suppressed by Mg2+. Several other E. coli nucleases, including exonucleases I, III, V, and VII, endonucleases I, III and IV and the 5'-->3' exonuclease function of DNA polymerase I, are unable to act as a dRpase. Nevertheless, E. coli fpg recJ double mutants retain capacity to repair abasic sites in DNA, indicating the presence of a back-up excision function.