scholarly journals Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide

1986 ◽  
Vol 235 (2) ◽  
pp. 531-536 ◽  
Author(s):  
M Dizdaroglu ◽  
E Holwitt ◽  
M P Hagan ◽  
W F Blakely
Keyword(s):  
Dna Repair ◽  
Formic Acid ◽  
Excision Repair ◽  
Dna Lesions ◽  
Gas Chromatography Mass Spectrometry ◽  
Dna Glycosylases ◽  
Thymine Glycol ◽  
Repair Enzymes ◽  
Base Excision

OsO4 selectively forms thymine glycol lesions in DNA. In the past, OsO4-treated DNA has been used as a substrate in studies of DNA repair utilizing base-excision repair enzymes such as DNA glycosylases. There is, however, no information available on the chemical identity of other OsO4-induced base lesions in DNA. A complete knowledge of such DNA lesions may be of importance for repair studies. Using a methodology developed recently for characterization of oxidative base damage in DNA, we provide evidence for the formation of cytosine glycol and 5,6-dihydroxycytosine moieties, in addition to thymine glycol, in DNA on treatment with OsO4. For this purpose, samples of OsO4-treated DNA were hydrolysed with formic acid, then trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. In addition to thymine glycol, 5-hydroxyuracil (isobarbituric acid), 5-hydroxycytosine and 5,6-dihydroxyuracil (isodialuric acid or dialuric acid) were identified in OsO4-treated DNA. It is suggested that 5-hydroxyuracil was formed by formic acid-induced deamination and dehydration of cytosine glycol, which was the actual oxidation product of the cytosine moiety in DNA. 5-Hydroxycytosine obviously resulted from dehydration of cytosine glycol, and 5,6-dihydroxyuracil from deamination of 5,6-dihydroxycytosine. This scheme was supported by the presence of 5-hydroxyuracil, uracil glycol and 5,6-dihydroxyuracil in OsO4-treated cytosine. Treatment of OsO4-treated cytosine with formic acid caused the complete conversion of uracil glycol into 5-hydroxyuracil. The implications of these findings relative to studies of DNA repair are discussed.

scholarly journals New perspectives in cancer biology from a study of canonical and non-canonical functions of base excision repair proteins with a focus on early steps

Mutagenesis ◽  
2019 ◽  
Vol 35 (1) ◽  
pp. 129-149 ◽  
Author(s):  
Matilde Clarissa Malfatti ◽  
Giulia Antoniali ◽  
Marta Codrich ◽  
Silvia Burra ◽  
Giovanna Mangiapane ◽  
...  
Keyword(s):  
Dna Repair ◽  
Base Excision Repair ◽  
Cancer Biology ◽  
Molecular Mechanisms ◽  
Excision Repair ◽  
Dna Lesions ◽  
Cancer Development ◽  
Dna Repair Enzymes ◽  
Repair Enzymes ◽  
Base Excision

Abstract Alterations of DNA repair enzymes and consequential triggering of aberrant DNA damage response (DDR) pathways are thought to play a pivotal role in genomic instabilities associated with cancer development, and are further thought to be important predictive biomarkers for therapy using the synthetic lethality paradigm. However, novel unpredicted perspectives are emerging from the identification of several non-canonical roles of DNA repair enzymes, particularly in gene expression regulation, by different molecular mechanisms, such as (i) non-coding RNA regulation of tumour suppressors, (ii) epigenetic and transcriptional regulation of genes involved in genotoxic responses and (iii) paracrine effects of secreted DNA repair enzymes triggering the cell senescence phenotype. The base excision repair (BER) pathway, canonically involved in the repair of non-distorting DNA lesions generated by oxidative stress, ionising radiation, alkylation damage and spontaneous or enzymatic deamination of nucleotide bases, represents a paradigm for the multifaceted roles of complex DDR in human cells. This review will focus on what is known about the canonical and non-canonical functions of BER enzymes related to cancer development, highlighting novel opportunities to understand the biology of cancer and representing future perspectives for designing new anticancer strategies. We will specifically focus on APE1 as an example of a pleiotropic and multifunctional BER protein.

scholarly journals Characterization of a Thermostable 8-Oxoguanine DNA Glycosylase Specific for GO/N Mismatches from the Thermoacidophilic ArchaeonThermoplasma volcanium

Archaea ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Miki Fujii ◽  
Chieri Hata ◽  
Munetada Ukita ◽  
Chie Fukushima ◽  
Chihiro Matsuura ◽  
...  
Keyword(s):  
Protein Gene ◽  
Excision Repair ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Oxygen Species ◽  
Dna Glycosylases ◽  
Optimal Activity ◽  
Base Excision ◽  
Ap Lyase

The oxidation of guanine (G) to 7,8-dihydro-8-oxoguanine (GO) forms one of the major DNA lesions generated by reactive oxygen species (ROS). The GO can be corrected by GO DNA glycosylases (Ogg), enzymes involved in base excision repair (BER). Unrepaired GO induces mismatched base pairing with adenine (A); as a result, the mismatch causes a point mutation, from G paired with cytosine (C) to thymine (T) paired with adenine (A), during DNA replication. Here, we report the characterization of a putative Ogg from the thermoacidophilic archaeonThermoplasma volcanium. The 204-amino acid sequence of the putative Ogg (TVG_RS00315) shares significant sequence homology with the DNA glycosylases ofMethanocaldococcus jannaschii(MjaOgg) andSulfolobus solfataricus(SsoOgg). The six histidine-tagged recombinant TVG_RS00315 protein gene was expressed inEscherichia coliand purified. The Ogg protein is thermostable, with optimal activity near a pH of 7.5 and a temperature of 60°C. The enzyme displays DNA glycosylase, and apurinic/apyrimidinic (AP) lyase activities on GO/N (where N is A, T, G, or C) mismatch; yet it cannot eliminate U from U/G or T from T/G, as mismatch glycosylase (MIG) can. These results indicate that TvoOgg-encodingTVG_RS00315is a member of the Ogg2 family ofT. volcanium.

scholarly journals Inhibition of DNA repair glycosylases by base analogs and tryptophan pyrolysate, Trp-P-1.

2005 ◽  
Vol 52 (1) ◽  
pp. 167-178 ◽  
Author(s):  
Elzbieta Speina ◽  
Jarosław M Cieśla ◽  
Maria-Anna Graziewicz ◽  
Jacques Laval ◽  
Zygmunt Kazimierczuk ◽  
...  
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Dna Structure ◽  
Dna Glycosylases ◽  
Ap Endonuclease ◽  
Abasic Sites ◽  
Repair Enzymes ◽  
Lyase Activity ◽  
Base Analog ◽  
Base Excision

DNA base analogs, 2,4,5,6-substituted pyrimidines and 2,6-substituted purines were tested as potential inhibitors of E. coli Fpg protein (formamidopyrimidine -DNA glycosylase). Three of the seventeen compounds tested revealed inhibitory properties. 2-Thioxanthine was the most efficient, inhibiting 50% of 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeG) excision activity at 17.1 microM concentration. The measured K(i) was 4.44 +/- 0.15 microM. Inhibition was observed only when the Fpg protein was first challenged to its substrate followed by the addition of the base analog, suggesting uncompetitive (catalytic) inhibition. For two other compounds, 2-thio- or 2-oxo-4,5,6-substituted pyrimidines, IC(50) was only 343.3 +/- 58.6 and 350 +/- 24.4 microM, respectively. No change of the Fpg glycosylase activity was detected in the presence of Fapy-7MeG, up to 5 microM. We also investigated the effect of DNA structure modified by tryptophan pyrolysate (Trp-P-1) on the activity of base excision repair enzymes: Escherichia coli and human DNA glycosylases of oxidized (Fpg, Nth) and alkylated bases (TagA, AlkA, and ANPG), and for bacterial AP endonuclease (Xth protein). Trp-P-1, which changes the secondary DNA structure into non-B, non-Z most efficiently inhibited excision of alkylated bases by the AlkA glycosylase (IC(50) = 1 microM). The ANPG, TagA, and Fpg proteins were also inhibited although to a lesser extent (IC(50) = 76.5 microM, 96 microM, and 187.5 microM, respectively). Trp-P-1 also inhibited incision of DNA at abasic sites by the beta-lyase activity of the Fpg and Nth proteins, and to a lesser extent by the Xth AP endonuclease. Thus, DNA conformation is critical for excision of damaged bases and incision of abasic sites by DNA repair enzymes.

scholarly journals Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

2021 ◽  
Vol 12 ◽  
Author(s):  
Almaz Nigatu Tesfahun ◽  
Marina Alexeeva ◽  
Miglė Tomkuvienė ◽  
Aysha Arshad ◽  
Prashanna Guragain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Excision Repair ◽  
Dna Lesions ◽  
Base Pairs ◽  
E Coli ◽  
Thymine Glycol ◽  
Base Excision ◽  
The Cost ◽  
Primary Substrate

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

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

2016 ◽  
Vol 113 (28) ◽  
pp. 7792-7797 ◽  
Author(s):  
Chenxu Zhu ◽  
Lining Lu ◽  
Jun Zhang ◽  
Zongwei Yue ◽  
Jinghui Song ◽  
...  
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Mammalian Genome ◽  
Dna Bases ◽  
Repair Pathway ◽  
Thymine Glycol ◽  
Base Excision ◽  
Oxidation Damage ◽  
Biochemical Analyses ◽  
Base Excision Dna Repair

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 enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.

scholarly journals Databases and Bioinformatics Tools for the Study of DNA Repair

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Kaja Milanowska ◽  
Kristian Rother ◽  
Janusz M. Bujnicki
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Dna Lesions ◽  
Environmental Chemicals ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Repair Mechanisms ◽  
Base Excision

DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER) and nucleotide excision repair (NER) or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS). There are also other mechanisms of DNA repair such as homologous recombination repair (HRR), nonhomologous end-joining repair (NHEJ), or DNA damage response system (DDR). This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions.

Response of Base Excision Repair Enzymes to Complex DNA Lesions

2001 ◽  
Vol 156 (5) ◽  
pp. 584-589 ◽  
Author(s):  
M. Weinfeld ◽  
A. Rasouli-Nia ◽  
M. A. Chaudhry ◽  
R. A. Britten
Keyword(s):  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Lesions ◽  
Repair Enzymes ◽  
Base Excision

scholarly journals The Base Excision Repair Pathway in the Nematode Caenorhabditis elegans

2020 ◽  
Vol 8 ◽  
Author(s):  
Noha Elsakrmy ◽  
Qiu-Mei Zhang-Akiyama ◽  
Dindial Ramotar
Keyword(s):  
Dna Repair ◽  
Caenorhabditis Elegans ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Glycosylases ◽  
Repair Synthesis ◽  
C Elegans ◽  
Base Excision ◽  
Dna Repair Synthesis

Exogenous and endogenous damage to the DNA is inevitable. Several DNA repair pathways including base excision, nucleotide excision, mismatch, homologous and non-homologous recombinations are conserved across all organisms to faithfully maintain the integrity of the genome. The base excision repair (BER) pathway functions to repair single-base DNA lesions and during the process creates the premutagenic apurinic/apyrimidinic (AP) sites. In this review, we discuss the components of the BER pathway in the nematode Caenorhabditis elegans and delineate the different phenotypes caused by the deletion or the knockdown of the respective DNA repair gene, as well as the implications. To date, two DNA glycosylases have been identified in C. elegans, the monofunctional uracil DNA glycosylase-1 (UNG-1) and the bifunctional endonuclease III-1 (NTH-1) with associated AP lyase activity. In addition, the animal possesses two AP endonucleases belonging to the exonuclease-3 and endonuclease IV families and in C. elegans these enzymes are called EXO-3 and APN-1, respectively. In mammalian cells, the DNA polymerase, Pol beta, that is required to reinsert the correct bases for DNA repair synthesis is not found in the genome of C. elegans and the evidence indicates that this role could be substituted by DNA polymerase theta (POLQ), which is known to perform a function in the microhomology-mediated end-joining pathway in human cells. The phenotypes observed by the C. elegans mutant strains of the BER pathway raised many challenging questions including the possibility that the DNA glycosylases may have broader functional roles, as discuss in this review.

scholarly journals Imbalanced Base Excision Repair Increases Spontaneous Mutation and Alkylation Sensitivity inEscherichia coli

1999 ◽  
Vol 181 (21) ◽  
pp. 6763-6771 ◽  
Author(s):  
Lauren M. Posnick ◽  
Leona D. Samson
Keyword(s):  
Base Excision Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Dna Lesions ◽  
Dna Glycosylases ◽  
E Coli ◽  
Abasic Sites ◽  
Base Excision ◽  
Harmful Effects

ABSTRACT Inappropriate expression of 3-methyladenine (3MeA) DNA glycosylases has been shown to have harmful effects on microbial and mammalian cells. To understand the underlying reasons for this phenomenon, we have determined how DNA glycosylase activity and substrate specificity modulate glycosylase effects in Escherichia coli. We compared the effects of two 3MeA DNA glycosylases with very different substrate ranges, namely, the Saccharomyces cerevisiae Mag1 and the E. coli Tag glycosylases. Both glycosylases increased spontaneous mutation, decreased cell viability, and sensitized E. coli to killing by the alkylating agent methyl methanesulfonate. However, Tag had much less harmful effects than Mag1. The difference between the two enzymes’ effects may be accounted for by the fact that Tag almost exclusively excises 3MeA lesions, whereas Mag1 excises a broad range of alkylated and other purines. We infer that the DNA lesions responsible for changes in spontaneous mutation, viability, and alkylation sensitivity are abasic sites and secondary lesions resulting from processing abasic sites via the base excision repair pathway.

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