Stereoselective excision of thymine glycol lesions by mammalian cell extracts

McTigue, Monica; Rieger, Robert; Rosenquist, Thomas A.; Iden, Charles R.; Santos, Carlos R. de los

doi:10.1016/j.dnarep.2003.11.009

Cited by 21 publications

(14 citation statements)

References 71 publications

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“…HeLa cell extracts or mouse ES cell nuclear extracts remove 5R-Tg considerably faster than 5S-Tg, and so the latter lesion might be more frequently encountered by a DNA replication fork (36,37). Use of POLN to bypass 5S-Tg with comparatively high efficiency and accuracy of bypass would be consistent with the low mutation frequency induced by Tg (38,39).…”

Section: Discussionmentioning

confidence: 79%

Human DNA Polymerase N (POLN) Is a Low Fidelity Enzyme Capable of Error-free Bypass of 5S-Thymine Glycol

Takata

Shimizu

Iwai

et al. 2006

Journal of Biological Chemistry

139

206

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Human DNA polymerase N (POLN or pol ) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiationinduced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.The human genome contains 15 distinct known DNA polymerase genes, and these are classified into four families A, B, X, and Y based on their amino acid sequences (1). Human DNA polymerase N (POLN), 2 the most recently discovered nuclear DNA polymerase, is an A-family enzyme with unknown function. The gene on chromosome 4p16.2 encodes a protein of 900 amino acid residues with a molecular mass of 100 kDa (2). The prototypical A-family DNA polymerase, Escherichia coli pol I is a high fidelity DNA polymerase that contributes to the maturation of Okazaki fragments during DNA replication and in gap-filling during base excision repair (BER), nucleotide excision repair (NER), and repair of DNA interstrand cross-links.Human POLQ, another A-family DNA polymerase, is similar to the Drosophila nuclear DNA polymerase Mus308 (3) in that it encodes both a DNA/RNA helicase domain and an A-family DNA polymerase domain (4, 5). By contrast, POLN has only the DNA polymerase domain. The POLN gene is encoded only in vertebrate genomes, but not in invertebrates or any lower eukaryotes. Possibly POLN has a role related to organ systems that are especially developed in vertebrates, such as the adaptive immune system or the brain. Expression studies of POLN are limited, but expression of the gene has been detected by Northern blotting in testes, heart and skeletal muscle tissue (2) and by expression sequence tagging in prostate, muscle, brain, and other organs. In cells from a human neuroblastoma patient, a chromosome fusion (1, 4) disrupting the DNA polymerase domain coding sequence of POLN was observed at diagnosis and at relapse. A (4, 17) fusion was detected at relapse only (6). It is possible that POLN might serve as a tumor suppressor in some cell types and that loss...

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Section: Discussionmentioning

confidence: 79%

Human DNA Polymerase N (POLN) Is a Low Fidelity Enzyme Capable of Error-free Bypass of 5S-Thymine Glycol

Takata

Shimizu

Iwai

et al. 2006

Journal of Biological Chemistry

139

206

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“…Besides the cis (5R, 6S) isomer used in this study, Tg can also exist in the other (5S, 6R) isomer; both diastereoisomers are efficiently removed by NEIL1 (41,42). In our structures, the two hydroxyl groups were recognized by water-mediated hydrogen bonds instead of direct interactions with the protein; such a feature might provide flexibility to the accommodation of both stereoisomers in the binding pocket.…”

Section: Discussionmentioning

confidence: 94%

Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

Zhu

Zhang

et al. 2016

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

119

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NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)-a preferred substrate-for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzymepromoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.base-excision repair | substrate recognition | enzyme catalysis | glycosylase | QM/MM D NA oxidation damage can be induced by both endogenous and environmental reactive oxygen species. Such oxidized DNA bases are primarily recognized and removed by the baseexcision repair (BER) pathway, which is initiated by a lesionspecific DNA glycosylase (1-4). Based on sequence homology and structural motifs, glycosylases that cleave oxidation damage are grouped into two families: the helix-hairpin-helix (HhH) family and the Fpg/Nei family (5, 6); the latter is named after the prototypical bacterial members formamidopyrimidine DNA glycosylase (Fpg) and endonuclease eight (Nei).NEIL1 (Nei-like 1) is one such Fpg/Nei family glycosylase that guards the mammalian genome against oxidation damage (7-10). NEIL1 is bifunctional in that it catalyzes both the hydrolysis of the N-glycosylic bond linking a base to a deoxyribose (glycosylase activity) and the subsequent cleavage of the DNA 3′ to the newly created apurinic/apyrimidinic site (lyase activity) (7-10). The N terminus contains the glycosylase domain of NEIL1, and the C terminus is intrinsically disordered (8,11). Whereas the C terminus is dispensable for both glycosylase and lyase activities in vitro, it interacts with many proteins in vivo and is required for efficient DNA repair activity inside the cells (12, 13). NEIL1 is also unique among the three human NEIL proteins in that it is increased in an S-phase-specific manner and carries out prereplicative repair of oxidized bases in the human genome (8,12). Moreover, increasing literature has further emphasized the importance of NEIL1's cellular repair activity, as NEIL1 deficiency has led to multiple abnormalities and is associated with severe human diseases, including cancer (14-19). Additionally, emerging evidence has also implicated a role of NEIL1 in active DNA demethylation (20)(21)(22).NEIL1 is capable of removing a wide array of oxidized pyrimidines and purines; representative substrates of extensive investigations include thymine glycol (Tg), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC), dihydrothymine (DHT), and dihydrouracil (DHU), as well as the formamidopyridines (FapyA and FapyG), spiroiminodihydantoin (Sp), and guanidinohydanto...

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“…It should be noted that cis-Tg is subject to slow interconversion at C6 to produce trans-Tg even in DNA (40), and, although the majority of these lesions in our experiments were cis-Tg (22), a small admixture of trans-Tg is likely, complicating the analysis of Nei stereospecificity. Whereas Tg has been shown to appear in vivo during oxidative stress (41), it is not clear whether DHU is formed in the cells, as this lesion is preferentially generated by ionizing radiation under oxygen-free conditions (26), and there is no direct assay for it in biological samples.…”

Section: Substrate Specificity Of Neimentioning

confidence: 88%

“…Duplex oligonucleotides containing a single AP site were obtained by treating the corresponding uracilcontaining duplexes with uracil-DNA glycosylase according to the manufacturer's instructions; the completeness of uracil excision was confirmed by heating the product with putrescine-HCl (pH 8.0) followed by polyacrylamide gel electrophoresis (PAGE) analysis (21). Synthesis and purification of oligonucleotides containing a single thymine glycol residue were performed as described previously (22). The sequence used in this case was 5′-GACAAGCGCAGYCAGCCGAACAC-3′, where Y was (5S,6R)-Tg or (5R,6S)-Tg.…”

Section: Enzymes and Oligonucleotidesmentioning

confidence: 99%

Catalytic Mechanism of Escherichia coli Endonuclease VIII: Roles of the Intercalation Loop and the Zinc Finger

2006

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Endonuclease VIII (Nei) excises oxidatively damaged pyrimidines from DNA and shares structural and functional homology with formamidopyrimidine-DNA glycosylase. Although the structure of Escherichia coli Nei is solved, the functions of many of its amino acid residues involved in catalysis and substrate specificity are not known. We constructed a series of Nei mutants that interfere with eversion of the damaged base from the helix (QLY69-71AAA, ΔQLY69-71) or perturb the conserved zinc finger (R171A, Q261A). Steady-state kinetics were measured with these mutant enzymes using substrates containing 5,6-dihydrouracil, two enantiomers of thymine glycol, 8-oxo-7,8-dihydroguanine and an abasic site positioned opposite each of the four canonical DNA bases. To some extent, all Nei mutants were deficient in processing damaged DNA, with mutations in zinc finger mutants generally having a more profound effect. Wild-type Nei showed prominent oppositebase specificity (G>C≈T>A) when the lesion was 5,6-dihydrouracil or cis-(5S,6R)-thymine glycol but not for other lesions tested. Mutations in the Q69-Y71 loop eliminated this effect. Only wildtype Nei and Nei-Q261A mutants could be reductively cross-linked to damaged base-containing DNA. Experiments involving trapping with NaBH 4 and the kinetics of DNA cleavage catalyzed by Nei-Q261A suggested that this mutant was deficient in regenerating free enzyme from the Nei-DNA covalent complex formed during the reaction. We conclude that the opposite-base specificity of Nei is primarily governed by residues in the Q69-Y71 loop and that both this loop and the zinc finger contribute significantly to the substrate specificity of Nei.Endonuclease VIII (Nei), a bacterial enzyme with DNA N-glycosylase and abasic site lyase (AP 1 lyase) activities, participates in base excision repair of oxidatively generated DNA damage (1,2). Nei is homologous to another prokaryotic enzyme, formamidopyrimidine-DNA glycosylase (Fpg) (3); together with Fpg and several eukaryotic homologs, it forms the Fpg/ Nei family of DNA repair glycosylases (4,5). Despite the structural homology, Nei preferentially removes oxidatively damaged pyrimidines from substrate DNA (1,2,6), whereas Fpg excises oxidatively damaged purines (7,8). During catalysis, the Nα of the N-terminal proline moiety of Nei attacks the C1' atom of the damaged nucleoside, leading to loss of the base and formation of a Schiff base intermediate. The latter rearranges to eliminate the 3′-phosphate (β-elimination) and 5′-phosphate (δ-elimination) from the deoxyribose moiety. In bacteria, Nei likely serves as a back-up for endonuclease III, the major glycosylase with activity against oxidatively damaged pyrimidines.* To whom correspondence should be addressed. D.O.Z.: tel. +7-383-335-6226, fax: +7-383-333-3677, email: dzharkov@niboch.nsc.ru. A.P.G.: tel. +1-631-444-3080, fax: +1-631-444-7641, email: apg@pharm.stonybrook NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe three-dimensional structure of Nei (9) and its coval...

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Stereoselective excision of thymine glycol lesions by mammalian cell extracts

Cited by 21 publications

References 71 publications

Human DNA Polymerase N (POLN) Is a Low Fidelity Enzyme Capable of Error-free Bypass of 5S-Thymine Glycol

Human DNA Polymerase N (POLN) Is a Low Fidelity Enzyme Capable of Error-free Bypass of 5S-Thymine Glycol

Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

Catalytic Mechanism of Escherichia coli Endonuclease VIII: Roles of the Intercalation Loop and the Zinc Finger

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