DNA glycosylase activities for thymine residues damaged by ring saturation, fragmentation, or ring contraction are functions of endonuclease III in Escherichia coli.

Breimer, Lars H.; Lindahl, Tomas

doi:10.1016/s0021-9258(18)91047-1

Cited by 243 publications

(81 citation statements)

References 39 publications

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“…Endonuclease III was prepared from Escherichia coli BW 420, an over-producer containing a nth gene (Cunningham & Weiss, 1985) in multicopy ColEl plasmid, that was kindly sent to us by B. Weiss and cultivated in the presence of colicin El as recommended. The purification was carried out up to fraction IV as described by Breimer & Lindahl (1984); the enzyme was followed by its nicking activity, destroyed by 5 min of heating at 60°C, on alkylated depurinated [3H]DNA (Paquette et al, 1972). The purified enzyme was kept at -20°C in 15 mM-potassium phosphate, pH 7.4, containing 0.5 M-NaCl, 1 mM-EDTA, 7 mM-2-mercaptoethanol and 5% (v/v) glycerol.…”

Section: Preparation Of E Coli Endonuclease Illmentioning

confidence: 99%

“…The target of endonuclease III is a thymine with a saturated 5,6 double bond; the enzyme is a DNA glycosylase that releases the modified thymine and also a 3' AP endonuclease that cuts the phosphodiester bond 3' to the AP site leaving a 3'-terminal deoxyribose that cannot be utilized by DNA polymerase I Warner et al, 1980). Katcher & Wallace (1983) and Breimer & Lindahl (1984) have completely purified endonuclease III. It has an Mr of 25000 and does not need any divalent cation to be active.…”

Section: Introductionmentioning

confidence: 99%

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

Bailly¹,

Verly²

1987

Biochemical Journal

248

181

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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.

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Section: Preparation Of E Coli Endonuclease Illmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Bailly¹,

Verly²

1987

Biochemical Journal

248

181

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“…These are termed as AP lyases that cleave 3 0 to AP sites using a b-elimination mechanism, leaving a 5 0phosphate group and a 3 0 -a,b-unsaturated aldehyde. The AP lyase activity is associated with bifunctional DNA glycosylases that are major repair enzymes existing in all organisms from bacteria to man (21)(22)(23)(24)(25)(26). The 3 0 -deoxyribose phosphate (3 0 -dRP) termini resulting from an AP lyase cleavage and other related 3 0 -blocking groups, such as 3 0 -phosphate and 3 0 -phosphoglycolate, inhibits DNA polymerases and can be converted into highly cytotoxic double strand breaks during replication (27).…”

Section: Introductionmentioning

confidence: 99%

A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

Alseth¹

2004

Nucleic Acids Research

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One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3'-blocking termini following AP lyase cleavage by Nth1.

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“…To address this question we placed thymine glycol (Tg), dihydrothymine (DHT) or an abasic site opposite a ssb and Tg or DHT opposite a purine lesion, 8-oxoG, and asked whether the presence of the lesion in the opposing strand affected recognition of the lesion in the target strand. All three target lesions, Tg, DHT and AP sites, are recognized by endos III (16) and VIII (11,17). The MDSs chosen are biologically relevant, since both Tg and DHT are radiolysis products of DNA thymine (18), the latter being formed only under anaerobic conditions, and 8-oxoG is a commonly produced purine lesion (19,20).…”

Section: Introductionmentioning

confidence: 99%

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII

Harrison

1998

Nucleic Acids Research

114

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Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.

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DNA glycosylase activities for thymine residues damaged by ring saturation, fragmentation, or ring contraction are functions of endonuclease III in Escherichia coli.

Cited by 243 publications

References 39 publications

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

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

A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII

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