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.     

Structure of Escherichia coli exonuclease I suggests how processivity is achieved

Exonuclease I (ExoI) from Escherichia coli is a monomeric enzyme that processively degrades single stranded DNA in the 3' to 5' direction and has been implicated in DNA recombination and repair. Determination of the structure of ExoI to 2.4 A resolution using X-ray crystallography verifies the expected correspondence between a region of ExoI and the exonuclease (or proofreading) domains of the DNA polymerases. The overall fold of ExoI also includes two other regions, one of which extends the exonuclease domain and another that can be described as an elaborated SH3 domain. These three regions combine to form a molecule that is shaped like the letter C, although it also contains a segment that effectively converts the C into an O-like shape. The structure of ExoI thus provides additional support for the idea that DNA metabolizing enzymes achieve processivity by completely enclosing the DNA.     

Stereochemical course of hydrolysis of DNA by exonuclease I from Escherichia coli

Exonuclease I has been purified from an overproducing strain of Escherichia coli K12 [Prasher, D. C., Conarro, L., & Kushner, S. R. (1983) J. Biol. Chem. 258, 6340-6343]. The enzyme hydrolyzes deoxyribonucleic acids that contain chiral phosphorothioate diester linkages, and the stereochemical course of the reaction is inversion of configuration at phosphorus. This result is most consistent with hydrolysis occurring via the direct attack of water on a phosphorothioate diester rather than through the intermediacy of a covalent nucleotidyl-enzyme intermediate. This finding represents the first example of a processive exonuclease whose stereochemical pathway has been determined.     

DNA deoxyribophosphodiesterase.

A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli. The enzyme catalyses the hydrolytic release of 2-deoxyribose-5-phosphate from single-strand interruptions in DNA with a base-free residue on the 5' side. The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5'-phosphatase activity. The enzyme has a mol. wt of approximately 50,000-55,000 and has been termed DNA deoxyribophosphodiesterase (dRpase). The protein presumably is active in DNA excision repair to remove a sugar-phosphate residue from an endonucleolytically incised apurinic/apyrimidinic site, prior to gap filling and ligation.     

Excision of sugar-phosphate products at apurinic/apyrimidinic sites by DNA deoxyribophosphodiesterase of Escherichia coli.

It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli. A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate. It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E. coli. The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM. It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione. The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases.     

Molecular structure of exonuclease I from Escherichia coli B

Exonuclease I of Escherichia coli B (EC 3.1.4.25) was determined to be a monomeric protein of molecular weight approx. 72,000, as estimated by Sephadex gel filtration, sedimentation velocity centrifugation, and sodium dodecylsulfate polyacrylamide gel electrophoresis.     

DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.

A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function.     

Deoxyribonucleic acid polymerase III of Escherichia coli. Characterization of associated exonuclease activities.

Purified DNA polymerase III has two distinct exonuclease activities: one initiates hydrolsis at the 3 termini, and the other at the 5 termini of single-stranded DNA. Both exonucleases have the same relative mobility on polyacrylamide gels as the polymerase activity. Molecular identity of the three activities is further indicated by their comparative rates of thermal inactivation and their sensitivity to ionic strength. The 3-5 exonuclease activity hydrolyzes only single-standed DNA. The rate of hydrolysis is twice the optimal rate of polymerization. The products are 5-mononucleotides, but the 3-5 activity is unable to cleave free dinucleotides or the 5-terminal dinucleotide of a polydeoxynucleotide chain. The 3-5 activity will not degrade 3-phosphoryl-terminated oligonucleotides such as d(pTpTpTp). The 5-3 activity catalyzes the hydrolysis of single-stranded DNA at 1/15 the rate of the 3-5 exonuclease. The 5-3 exonuclease requires the presence of a 5 single-stranded terminus in order to initiate hydrolysis, but will thereafter proceed into a double-stranded region. Although the limit products found during hydrolysis of substrates designed to assay specifically the 5-3 activity are predominantly mono- and dinucleotides, these products probably arise from the subsequent hydrolysis of oligonucleotides by the 3-5 hydrolytic activity. This interpretation is supported by (a) the relatively greater activity of the 3-5 exonuclease, (b) the inability of the enzyme to degrade d(pTpTpTp), and (c) the release of the 5 terminus of a single-stranded DNA molecule as an oligonucleotide. The 5-3 exonuclease attacks ultraviolet-irradiated duplex DNA which has first been incised by the Micrococcus luteus endonuclease specific for thymine dimers in DNA.     

Novel properties of Escherichia coli exonuclease III.

The specificity of hydrolysis of polynucleotide termini by Escherichia coli exonuclease III was studied with the use of oligothymidylate annealed to polydeoxyadenylate. The size of the products after 3' leads to 5'-hydrolysis of 5'-labeled substrate is temperature-dependent. At 25 degrees the enzyme can hydrolyze a polynucleotide chain up to the last 5'-terminal dinucleotide. A gradation of higher 5'-terminal oligonucleotides of defined chain lengths is produced after limit digestion by the enzyme when the temperature is raised between 25 degrees to 60 degrees. When the oligothymidylate was labeled at the 3'-ends with ribonucleotides, it was observed that exonuclease III can cleave a single or two consecutive ribonucleotides regardless of whether the ribonucleotides are base-paired or mismatched.     

Mutagenic DNA repair in Escherichia coli. XIII. Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis

We have introduced a mutD5 mutation (which results in defective 3'-5'-exonuclease activity of the epsilon proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect on UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the epsilon proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

Excision of gamma-ray damaged thymine by E. coli extracts is due to the 5'leads to 3' exonuclease associated with DNA polymerase I

Extracts of $\text{E. coli pol}$Aexl which contains a temperature sensitive 5′→3′ exonuclease function of polymerase I accomplish the selective excision of products of the 5,6-dihydroxy-dihydrothymine type from γ-irradiated DNA and OsO₄-oxidized polyd(A-T) at the permissive temperature (30°) but not at the nonpermissive temperature (42°). The 5′→3′ exonuclease activity of polymerase I, therefore, acts as a repair exonuclease in γ-ray excision repair.     

Escherichia coli mutants deficient in exonuclease VII.

Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains.     

Amplification and purification of exonuclease I from Escherichia coli K12

Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities. The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M. (1974) J. Biol. Chem. 249, 5379-5381). Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.     

Processivity and kinetics of the reaction of exonuclease I from Escherichia coli with polydeoxyribonucleotides

The enzyme exonuclease I from Escherichia coli hydrolyzes successive nucleotides from the 3'-termini of single-stranded deoxyribonucleotide homopolymers. When the reaction is stopped after partial hydrolysis, only intact starting material and small oligomers can be isolated. The distribution of oligomeric products varies with the base composition of the polymer but the largest oligomer that can be isolated from the reaction of exonuclease I with homopolymers of deoxyadenylate, deoxythymidylate, or deoxycytidylate is a decamer. These results suggest a model in which exonuclease I possesses at least two nucleotide binding sites. When both sites are filled, with 11-mers and longer polymers, the enzyme does not dissociate from the polymer during hydrolysis. When, with smaller oligomers, only a single site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation. The kinetics of the reactions of exonuclease I with purified polydeoxyriboadenylates of defined size distributions have been investigated. The maximum rates of hydrolysis are nearly independent of polymer size while the apparent Michaelis constants are inversely proportional to the polymer size. A simple steady state model yields a kinetic equation that is consistent with our results. Competition experiments indicate that the rate at which exonuclease I associates with the 3'-terminus of a polydeoxyribonucleotide is independent of the polymer's chain length.     

Mutants of Escherichia coli with altered deoxyribonucleases. II. Isolation and characterization of mutants for exonuclease I

Mutants of Escherichia coli K12 deficient in exonuclease I (xon^?)³ were identified by enzymic assay of randomly selected, heavily mutagenized clones. From one of the six mutants of independent origin a thermolabile variant of exonuclease I was partially purified and identified, indicating that the mutation is probably in a structural gene for the enzyme. Transduction of this mutation into a recB^? recC^? strain did not result in the suppression of any of the phenotypic traits of the recipient. Although the five other mutants also appear to have temperature-sensitive exonuclease I activities in crude extracts, these enzymes were not sufficiently stable to permit purification. These latter mutations were of the xonA^? type; they produced a temperature-dependent suppression of the sensitivity to ultraviolet light and to mitomycin C manifested by a recB^? recC^? strain. None of the six mutations were of the sbcB^? type; that is, they did not suppress the recombination deficiency of a recB^? recC^? strain.In experiments with bacteriophage Plke, the six mutations were 41 to 62% cotransducible with the his region of E. coli. Heterozygous F′-merodiploids were constructed and studied for possible complementation of exonuclease I activity. All six mutations and an sbcB^? mutation were recessive to the wild-type alleles, and all were found to belong to a single complementation group. The results suggest that alterations of a structural gene for exonuclease I may result in the indirect suppression of the ultraviolet and mitomycin sensitivity manifested by recB^? recC^? strains.     

Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion

Escherichia coli Exonuclease I (ExoI) digests single-stranded DNA (ssDNA) in the 3′-5′ direction in a highly processive manner. The crystal structure of ExoI, determined previously in the absence of DNA, revealed a C-shaped molecule with three domains that form a central positively charged groove. The active site is at the bottom of the groove, while an extended loop, proposed to encircle the DNA, crosses over the groove. Here, we present crystal structures of ExoI in complex with four different ssDNA substrates. The structures all have the ssDNA bound in essentially the predicted manner, with the 3′-end in the active site and the downstream end under the crossover loop. The central nucleotides of the DNA form a prominent bulge that contacts the SH3-like domain, while the nucleotides at the downstream end of the DNA form extensive interactions with an ‘anchor’ site. Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation. The highest-resolution structure, determined at 1.95 Å, reveals an Mg²⁺ ion bound to the scissile phosphate in a position corresponding to Mg^B in related two-metal nucleases. The structures provide new insights into the mechanism of processive digestion that will be discussed.