Characterization of a cross-linked DNA-Endonuclease VIII repair complex by electrospray ionization mass spectrometry

Rieger, Robert; McTigue, Monica; Kycia, Jadwiga H.; Gerchman, Sue Ellen; Grollman, Arthur P.; Iden, Charles R.

doi:10.1016/s1044-0305(00)00117-3

Cited by 51 publications

(41 citation statements)

References 20 publications

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“…Four selenium sites were located by using CNS (44), including that coming from the substituted N-terminal formylmethionine (fMet). Because the Fpg͞Nei family members require processing of the N-terminal fMet to use the second proline residue for enzyme catalysis (13)(14)(15)45), we expected to find only three selenium sites. A glycosylase assay revealed that SeMet-NEIL1C⌬56 exhibited a reduced activity on a 5,6-dihydrouracilcontaining substrate (data not shown), suggesting partial or incomplete processing of N-terminal fMet in the SeMet enzyme.…”

Section: Methodsmentioning

confidence: 99%

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The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

Doublié

Bandaru

Bond

et al. 2004

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

136

194

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In prokaryotes, two DNA glycosylases recognize and excise oxidized pyrimidines: endonuclease III (Nth) and endonuclease VIII (Nei). The oxidized purine 8-oxoguanine, on the other hand, is recognized by Fpg (also known as MutM), a glycosylase that belongs to the same family as Nei. The recent availability of the human genome sequence allowed the identification of three human homologs of Escherichia coli Nei. We report here the crystal structure of a human Nei-like (NEIL) enzyme, NEIL1. The structure of NEIL1 exhibits the same overall fold as E. coli Nei, albeit with an unexpected twist. Sequence alignments had predicted that NEIL1 would lack a zinc finger, and it was therefore expected to use a different DNA-binding motif instead. Our structure revealed that, to the contrary, NEIL1 contains a structural motif composed of two antiparallel ␤-strands that mimics the antiparallel ␤-hairpin zinc finger found in other Fpg͞Nei family members but lacks the loops that harbor the zinc-binding residues and, therefore, does not coordinate zinc. This ''zincless finger'' appears to be required for NEIL1 activity, because mutating a very highly conserved arginine within this motif greatly reduces the glycosylase activity of the enzyme.

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Section: Methodsmentioning

confidence: 99%

“…6 and 12). Catalysis by both E. coli Nei (EcoNei) and Fpg (EcoFpg) is by means of the N-terminal proline, which forms a Schiff base with the oxidized lesion (13)(14)(15). Both EcoNei and EcoFpg are trifunctional enzymes containing glycosylase, ␤,␦ lyase, and 5Ј phosphodiesterase activities (16)(17)(18)(19).…”

mentioning

confidence: 99%

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

Doublié

Bandaru

Bond

et al. 2004

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

136

194

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“…As a result, the enzyme and DNA form an imino Schiff base intermediate, and this protein-DNA complex can be covalently trapped in the presence of a reducing agent, such as NaBH 4 . Both endoVIII and Fpg use the secondary amine of their N-terminal proline residues as the nucleophile (21,22). In addition to an active site nucleophile, glycosylase/ lyase enzymes require acidic side chains to perform catalysis on damaged bases.…”

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confidence: 99%

Determination of Active Site Residues in Escherichia coli Endonuclease VIII

Burgess¹,

Jaruga²,

Dodson³

et al. 2002

Journal of Biological Chemistry

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Endonuclease VIII from Escherichia coli is a DNA glycosylase/lyase that removes oxidatively damaged bases. EndoVIII is a functional homologue of endonuclease III, but a sequence homologue of formamidopyrimidine-DNA glycosylase (Fpg). Using multiple sequence alignments, we have identified six target residues in endoVIII that may be involved in the enzyme's glycosylase and/or lyase functions: the N-terminal proline, and five acidic residues that are completely conserved in the endoVIIIFpg proteins. To investigate the contribution of these residues, site-directed mutagenesis was used to create seven mutants: P2T, E3D, E3Q, E6Q, D129N, D160N, and E174Q. Each mutant was assayed both for lyase activity on abasic (AP) sites and for glycosylase/lyase activity on 5-hydroxyuracil, thymine glycol, and ␥-irradiated DNA with multiple lesions. The P2T mutant did not have lyase or glycosylase/lyase activity but could efficiently form Schiff base intermediates on AP sites. E6Q, D129N, and D160N behaved essentially as endoVIII in all assays. E3D, E3Q, and E174Q retained significant AP lyase activity but had severely diminished or abolished glycosylase/lyase activities on the DNA lesions tested. These studies provide detailed predictions concerning the active site of endoVIII.Oxidative damage to cellular DNA results from its interaction with free radical species, which can originate from endogenous aerobic respiration or from exogenous free radical-generating agents. Oxidation of bases results in a variety of damages, many of which are potentially mutagenic; additionally, oxidative DNA damage has been implicated in aging and diseases, such as cancer (1-4). Base excision repair (BER) 1 is the primary pathway by which oxidative DNA damage is repaired, and this process is initiated by removal of the damaged base by a DNA glycosylase (5). In Escherichia coli, there are three known BER glycosylases that recognize and remove oxidized bases. Formamidopyrimidine-DNA glycosylase (Fpg) primarily removes oxidized purines such as 7,8-dihydro-8-oxoguanine (OG) and formamidopyrimidines (6, 7). Endonuclease III (endoIII) is the glycosylase primarily responsible for removal of oxidized pyrimidines, such as thymine glycol, 5,6-dihydrouracil, and 5,6-dihydrothymine (8, 9). The third E. coli BER glycosylase that removes oxidized bases is endonuclease VIII (endoVIII) (10, 11). EndoVIII has highly overlapping substrate specificity with endoIII, as evidenced through biochemical analyses (12) and the analyses of mutator phenotypes of E. coli strains deficient in one or both of these enzymes (11, 13). Although cells lacking functional genes for either endoIII (nth gene) or endoVIII (nei gene) have a weak mutator phenotype (13, 14), cells lacking both glycosylases have a much higher spontaneous mutation frequency (11) and a greater H 2 O 2 sensitivity (13).There is evidence that endoVIII also has slight substrate overlap with Fpg. Studies using oligonucleotides with sitespecific damage have shown that there are common substrates between the two enzymes...

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“…To form the duplex, 80 M 30-mer and 160 M 25-mer were incubated in water for 2 min at 95°C followed by 5 min at 37°C and 30 min at room temperature. E. coli formamidopyrimidine-DNA glycosylase (Fpg) and Nei proteins were purified as described previously (31,32). Purification of E. coli Nth will be described elsewhere.…”

Section: Methodsmentioning

confidence: 99%

“…Analytical Cross-linking-Cross-linking of Fpg, Nei, and Nth for use as standards was performed as described earlier (31,33) except for the different substrate sequence. For cell extracts, the reaction mixture (10 l) contained the thawed extract (8 l), 50 nM 32 P-labeled oligonucleotide duplex, and 50 mM NaBH 4 .…”

Section: Methodsmentioning

confidence: 99%

Proteomic Approach to Identification of Proteins Reactive for Abasic Sites in DNA

Rieger

Zaika

Xie

et al. 2006

Molecular & Cellular Proteomics

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Apurinic/apyrimidinic (AP) sites, a prominent type of DNA damage, are repaired through the base excision repair mechanism in both prokaryotes and eukaryotes and may interfere with many other cellular processes. A full repertoire of AP site-binding proteins in cells is presently unknown, preventing reliable assessment of harm inflicted by these ubiquitous lesions and of their involvement in the flux of DNA metabolism. We present a proteomics-based strategy for assembling at least a partial catalogue of proteins capable of binding AP sites in DNA. The general scheme relies on the sensitivity of many AP site-bound protein species to NaBH 4 cross-linking. An affinity-tagged substrate is used to facilitate isolation of the cross-linked species, which are then separated and analyzed by mass spectrometry methods. We report identification of seven proteins from Escherichia coli 6 bases in mammalian cells) measured by modification with a specific reagent in situ (4). Regardless of which estimate is more plausible, it is clear that AP sites represent a prominent type of DNA damage. AP sites may be unequally distributed over the genome (5), and their number increases greatly under challenging conditions such as x-ray irradiation (6). They are intrinsically unstable and can be converted to single strand breaks by heat, chemical bases, nucleophiles, or magnesium ions (1, 7). AP sites are highly mutagenic in vivo (8).Normally AP sites are repaired through the base excision repair (BER) mechanism in both prokaryotes and eukaryotes. A group of dedicated enzymes, AP endonucleases, is responsible for safeguarding the genome against these lesions (1). AP endonucleases hydrolyze DNA 5Ј to an AP site, leaving 3Ј-hydroxyl and 5Ј-phosphate termini at the nick; such an intermediate is then processed through either short patch or long patch BER subpathways with the involvement of different DNA polymerases, DNA ligases, and accessory factors (9). AP sites can also be cleaved through ␤-elimination by BER enzymes of another class, DNA glycosylases with an associated AP lyase activity (1, 9). In this case, an ␣,␤-unsaturated aldehyde is formed at the 3Ј-end, and a phosphate is formed at the 5Ј-end of DNA at the nick that is further processed by AP endonuclease and the downstream BER enzymes. During AP site cleavage by AP lyase activity of DNA glycosylases, an aldimine intermediate is formed between an active site amine of the protein and the C-1Ј atom of the baseless deoxyribose (10, 11). This intermediate, usually referred to as a "Schiff base," is easily reduced with sodium borohydride or related compounds, forming a stable amino link between the enzyme and DNA; this reaction is widely used to prove the ␤-elimination reaction mechanism for the enzymes capable of AP site cleavage (12). Some DNA glycosylases (e.g. Escherichia coli MutY protein) can form a Schiff base with no further ␤-elimination (13), but their affinity for the AP site is still very high.In addition to being a subject for recognition by specific DNA repair enzymes, AP site...

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Characterization of a cross-linked DNA-Endonuclease VIII repair complex by electrospray ionization mass spectrometry

Cited by 51 publications

References 20 publications

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

Determination of Active Site Residues in Escherichia coli Endonuclease VIII

Proteomic Approach to Identification of Proteins Reactive for Abasic Sites in DNA

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