<i>Escherichia coli</i> endonuclease III is not an endonuclease but a <i>β</i>-elimination catalyst

Bailly, Véronique; Verly, Walter G.

doi:10.1042/bj2420565

Cited by 248 publications

(198 citation statements)

References 18 publications

(17 reference statements)

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“…With the purified proteins, the end group termini (data not shown) are consistent with the AP lyase activity of Nth involving a ␤-elimination process as described by Bailly and Verly (35). The 3Ј OH end termini produced by cleavage of AP sites by the nuclear extract are consistent with the clean up of the end termini.…”

Section: Discussionsupporting

confidence: 85%

Clustered DNA Damage, Influence on Damage Excision by XRS5 Nuclear Extracts and Escherichia coli Nth and Fpg Proteins

David-Cordonnier¹,

Laval²,

O’Neill³

2000

Journal of Biological Chemistry

165

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Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damage, which are thought to reflect the biological severity. Escherichia coli Nth and Fpg and nuclear extracts from XRS5, a Chinese hamster ovary Ku-deficient cell line, have been used to study the influence on their substrate recognition by the presence of a neighboring damage or an abasic site on the opposite strand, as models of clustered DNA damage. These proteins were tested for their efficiency to induce a single-strand break on a 32 P-labeled oligonucleotide containing either an abasic (AP) site, dihydrothymine (DHT), 7,8-dihydro-8-oxo-2deoxygua-nine, or 7,8-dihydro-8-oxo-2deoxyadenine at positions 1, 3, or 5 base pairs 5 or 3 to either an AP site or DHT on the labeled strand. DHT excision is much more affected than cleavage of an AP site by the presence of other damage. The effect on DHT excision is greatest with a neighboring AP site, with the effect being asymmetric with Nth and Fpg. Therefore, this large inhibition of the excision of DHT by the presence of an opposite AP site may minimize the formation of double-strand breaks in the processing of DNA clustered damages.Radiation and radiation mimetic anticancer agents cause DNA damage, and it is thought that clustered damage (in which at least two damages are produced within less than 10 base pairs) is implicated in the biological severity of radiation since it is less repairable (1, 2). The complexity of radiationinduced clustered DNA damage increases on increasing the ionizing density of the radiation (LET).1 From track structure simulations, ϳ20% of double-strand breaks are associated with other damages for low LET radiation but increased to Ͼ20% for double-strand breaks induced by high LET ␣-radiation (3). Indirect experimental evidence supporting the role of DNA damage complexity comes from the reduced repairability of double-strand breaks induced in cellular DNA by high LET radiation (4, 5) and the increased complexity of single-strand breaks on increasing radiation quality as revealed using cell extracts (6 -9). If base damages within a cluster are on opposite strands and both excised, this gives rise to double-strand breaks. Therefore, it is of great significance to understand the way in which several damages in close proximity are recognized/processed by the cell.Although the chemical nature of oxidative damage produced by oxidative stress and by ionizing radiation are similar, the unique feature of ionizing radiation and radiation mimetic agents is their ability to produce clustered damage. There are only few studies about the excision of a damage substrate in the vicinity of another damage. In particular, synthesized oligonucleotides containing damage at specific sites were used to focus on the efficiencies of endonucleases VIII (Nei) and III (Nth) to excise either thymine glycol or DHT when opposite a singlestrand gap (10) as well as the efficiency of Fpg to excise 8-oxo-G or AP site opposite a gap (11) or 8-oxo-G near a formylamine on the same strand (12). Chaudhry...

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Section: Discussionsupporting

confidence: 85%

Clustered DNA Damage, Influence on Damage Excision by XRS5 Nuclear Extracts and Escherichia coli Nth and Fpg Proteins

David-Cordonnier¹,

Laval²,

O’Neill³

2000

Journal of Biological Chemistry

165

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“…6A, lane 4), the result of a ␤-elimination that leaves an ␣,␤-unsaturated aldehyde at the 3Ј terminus of the nucleoside gap. The additional minor bands are presumably due to isomerization of the 3Ј-hydroxypentenal terminus (33). Incubation of the 5-meC substrate with DME during 60 min generated a fragment comigrating with the AtOGG1 ␤-elimination product (Fig.…”

Section: Dme and Ros1 Process 5-mec Through A Dna Glycosylase͞lyasementioning

confidence: 98%

DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases

Morales‐Ruíz

Ortega-Galisteo

Ponferrada-Marín

et al. 2006

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

292

270

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Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase͞lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants.Arabidopsis ͉ demethylation ͉ gene silencing ͉ methylation M ethylation of cytosine at carbon 5 of the pyrimidine ring [5-methylcytosine (5-meC)] is an epigenetic modification that guides formation of transcriptionally silent chromatin and allows transmission of specific patterns of gene activity across cellular divisions (1, 2). In eukaryotes, DNA methylation is detected in protists, fungi, plants, and animals (3) and plays important roles in the establishment of developmental programs (4, 5) and in genome defense against parasitic mobile elements (6). Most of mammalian and plant DNA methylation is restricted to symmetrical CpG sequences, but plants also have significant levels of cytosine methylation in the symmetric context CpNpG (where N is any nucleotide) and even in asymmetric contexts (2, 7). Similarly to other biochemical modifications such as protein phosphorylation and acetylation, DNA methylation is also reversible. Demethylation may take place as a passive process because of lack of maintenance methylation during several cycles of DNA replication or as an active mechanism in the absence of replication (8). In mammalian preimplantation embryos the maternal genome is demethylated by a passive process along cleavage stages, whereas the paternal genome is demethylated by an active mechanism immediately after fertilization (9). In addition to global demethylation, site-specific...

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“…Thus, despite their structural similarities, these DNA repair enzymes have different substrate specificity. The catalytic mechanism of endonuclease III involves the glycosylase activity, followed by the ␤-elimination reaction which cleaves the phosphodiester bond 3Ј to an AP site (7). Results indicate that the M. luteus pyrimidine dimer glycosylase also follows a similar reaction mechanism (2).…”

mentioning

confidence: 99%

Identification of the Structural and Functional Domains of MutY, an DNA Mismatch Repair Enzyme

Manuel

Czerwinski

Lloyd

1996

Journal of Biological Chemistry

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The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26-and 13-kDa fragments. NH 2 -terminal sequencing showed that the p13 domain begins at Gln 226 , indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A⅐G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met 1 -Trp 216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.Proteins are constructed on a modular basis, and frequently these modules or domains are known to have unique functions. Isolation and characterization of these domains provide significant insight into the relationship between particular structural elements of the enzyme and its various activities. The mutY gene of Escherichia coli encodes a 39.1-kDa DNA mismatch repair protein. A significant portion of this protein is homologous to the 26.3-kDa E. coli endonuclease III. These two proteins are 66.3% similar and 23.8% identical over a 181-amino acid region (1). Another enzyme with sequence similarity to MutY is the product of the pdg gene in Micrococcus luteus, which recognizes and incises DNA containing cyclobutane pyrimidine dimers (2). The three enzymes mentioned above contain a [4Fe-4S] 2ϩ cluster, coordinated by four cysteine residues which are perfectly conserved in all three proteins.In contrast to their structural similarities, the functional properties of the above three DNA repair proteins are very different. MutY recognizes and removes the undamaged adenine mispaired with guanine, cytosine, 8-oxo-7,8-dihydro-2Ј-deoxyguanine (8-oxo-dG) 1 and 8-oxo-7,8-dihydro-2Ј-deoxyadenine (3) and is also reported to have AP endonuclease activity (4, 5). In vitro experiments show that MutY also recognizes and removes adenine analogs when they are paired with guanine (5).2 Endonuclease III primarily removes oxidized pyrimidines from DNA (6), and the pdg gene product in M. luteus repairs UV-induced pyrimidine dimer lesions in DNA (2). Thus, despite their structural similarities, these DNA repair enzymes have different substrate specificity. The catalytic mechanism of endonuclease III in...

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

Cited by 248 publications

References 18 publications

Clustered DNA Damage, Influence on Damage Excision by XRS5 Nuclear Extracts and Escherichia coli Nth and Fpg Proteins

Clustered DNA Damage, Influence on Damage Excision by XRS5 Nuclear Extracts and Escherichia coli Nth and Fpg Proteins

DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases

Identification of the Structural and Functional Domains of MutY, an DNA Mismatch Repair Enzyme

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