Properties of the Main Endonuclease Specific for Apurinic Sites of <i>Escherichia coli</i> (Endonuclease VI)

Gossard, Françis; Verly, Walter G.

doi:10.1111/j.1432-1033.1978.tb12026.x

Cited by 91 publications

(54 citation statements)

References 26 publications

(8 reference statements)

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“…We found that the exonuclease III-induced nicking of urea-containing PM2 DNA was dependent on Mg2", with optimal activity between 2 and 12 mM MgCl2 (data not shown). A similar Mg2' requirement was observed when AP DNA was used as the substrate (17).…”

supporting

confidence: 66%

Exonuclease III recognizes urea residues in oxidized DNA.

Kow¹,

Wallace²

1985

Proc. Natl. Acad. Sci. U.S.A.

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Escherichia coli exonuclease Im was found to be associated with an activity that recognizes urea residues in DNA but not thymine glycol residues from which the urea residues were prepared. This activity was not due to a contaminating activity such as endonuclease m since ureacontaining DNA was a competitive inhibitor of exonuclease III when apurinic DNA was used as a substrate and vice versa. The apparent kinetic constants for both the substrate and inhibitor were determined. Like its apurinic activity, exonuclease mI activity against urea residues was endonucleolytic, nicking on the 5' side of the damage and having an optimal Mg2" concentration between 2 and 10 mM. Also, the enzyme recognized alkali-stable damages produced in DNA by H202 in vitro. We suggest that it may be this activity of exonuclease III that accounts for its biological role in vivo.Exonuclease III ofEscherichia coli is a complex enzyme that contains 3' exonuclease and phosphatase activities specific for duplex DNA (1, 2) as well as an apurinic (AP) endonucleolytic activity (3-5). Even though the AP activity of exonuclease III accounts for about 90% of the total AP activity in the E. coli cell (6, 7), xth mutants that lack exonuclease III (8) are not markedly sensitive to alkylating agents, such as methyl methanesulfonate, that produce AP sites (6, 7). Further, single-strand breaks produced by ionizing radiation may contain 3' phosphate termini or 3' glycolic ends (9). Such termini would require a 3' phosphataseexonuclease activity so that the strand breaks would be a substrate for repair by DNA polymerase I. Although exonuclease III accounts for >99% of this activity in E. coli (10), xth mutants are not sensitive to x-rays (7).The most profound phenotype thus far associated with exonuclease III deficiency is the extreme sensitivity of xth mutants to H202, about 20-fold greater than that of wild type (11). We report here that exonuclease III can endonucleolytically incise adjacent to alkali-stable fragmented residues of thymine in DNA, and we propose that this activity may be responsible for repairing DNA damage produced by free radicals generated by metabolic oxygen and that this may be the principal biological role for exonuclease III. Polyscience (Warrington, PA). MATERIALS AND METHODSPreparation of Substrates. For preparation of thymine glycol-containing PM2 DNA, PM2 DNA was preheated at 650C for 2-5 min and then brought to 0.04% OS04 from a 4% stock. The DNA was incubated for another 5 min, quickly cooled on ice, and then dialyzed extensively to remove the OSO4. The treatment produced approximately one thymine glycol per DNA molecule. The number ofthymine glycols per DNA molecule was determined after exhaustive digestion with endonuclease III (13). The number of endonuclease III-sensitive sites produced under these conditions is about half the number of 5,6-dihydroxydihydrothymine-type damages, as determined by the acetol fragment assay (13), and is equal to the number of anti-thymine glycol antibody sites (unpublished data). To p...

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supporting

confidence: 66%

Exonuclease III recognizes urea residues in oxidized DNA.

Kow¹,

Wallace²

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…After incubation for 30 min at 37°C, the DNA was precipitated with ethanol (25) and dissolved in 10 mM Tris buffer (pH 8.0)-i mM trisodium EDTA. The substrates were tested by measuring the release of acid-soluble material (measured as for (15), and the preparation was dialyzed against 10 mM potassium phosphate buffer (pH 7.4) containing 1 mM trisodium EDTA. Four percent of the reduced DNA was alkali labile.…”

Section: Methodsmentioning

confidence: 99%

Endonuclease IV (nfo) mutant of Escherichia coli

Cunningham¹,

Saporito²,

Spitzer³

et al. 1986

J Bacteriol

366

278

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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, H202, 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 (23) of Escherichia coli is an apurinicapyrimidinic (AP) DNA endonuclease, i.e., a DNase specific for apurinic and apyrimidinic sites in DNA. It catalyzes the cleavage of a phosphodiester bond 5' to the AP site and is an example of the most prevalent form of AP endonuclease found throughout nature. Within E. coli, however, the enzyme is a minor one; it accounts for no more than 10% of the AP endonucleolytic activity measured in crude extracts (24). The major AP endonucleolytic activity of E.

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“…An important aspect of the action pattern of these enzymes is that they are unable to catalyze the liberation of free dRp from incised AP sites. Instead, they release dRp as part of a small oligonucleotide (15,16,39). Moreover, they do so at a reduced rate compared with excision of undamaged 5' nucleotide residues at nicks in DNA (35,39).…”

mentioning

confidence: 99%

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

Dianov¹,

Price²,

Lindahl³

1992

Mol. Cell. Biol.

274

187

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The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase 13.DNA bases altered by hydrolytic deamination, oxygen radical-induced damage, and alkylation are often excised by DNA glycosylases (44,54). Certain mismatched residues, such as adenine or thymine opposite guanine, may also be removed by specific DNA glycosylases (1, 56, 57). Thus, a common intermediate in the repair of several different types of DNA damage is an apurinic-apyrimidinic (AP) site. Such sites are also generated by nonenzymatic hydrolysis of base-sugar bonds, in particular by depurination (26), and as a consequence of exposure to ionizing radiation (12,27,34). AP endonucleases, for example, Escherichia coli exonuclease III and endonuclease IV, rapidly incise DNA at the 5' side of an AP site (9). Subsequently, an excision step occurs to generate a small gap, which is filled in by repair replication. A DNA ligase completes the repair process. These consecutive events take place rapidly and efficiently in vivo. It has been shown that AP sites occur transiently in DNA after X-irradiation of mammalian cells but disappear in 2 to 4 min without any detectable accumulation of DNA containing single-strand interruptions (34).The excision of a depurinated-depyrimidinated site has remained the least understood of these events in the base excision repair process. Early work by Painter and Young (37) and Regan and Setlow (40) showed that repair replication of X-irradiated and alkylated DNA in vivo involved the filling-in of much smaller gaps than those observed after UV light-induced DNA damage. Subsequent studies have also indicated that patches only 1 to 4 nucleotides long may be generated during repair of AP sites (8,32). The excision of a 5'-terminal base-free sugar-phosphate residue, possibly accompanied by the removal of one or a few adjacent residues, has been ascribed to exonuclease acti...

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Properties of the Main Endonuclease Specific for Apurinic Sites of Escherichia coli (Endonuclease VI)

Cited by 91 publications

References 26 publications

Exonuclease III recognizes urea residues in oxidized DNA.

Exonuclease III recognizes urea residues in oxidized DNA.

Endonuclease IV (nfo) mutant of Escherichia coli

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

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