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 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.

Requirements for DNA bubble structure for efficient cleavage by helix–two-turn–helix DNA glycosylases

Mutagenesis ◽  
2019 ◽  
Vol 35 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Kristina A Makasheva ◽  
Anton V Endutkin ◽  
Dmitry O Zharkov
Keyword(s):  
Excision Repair ◽  
Dna Lesions ◽  
Telomere Maintenance ◽  
Oxygen Species ◽  
Dna Glycosylases ◽  
The Third ◽  
Double Stranded Dna ◽  
Bubble Length ◽  
Bubble Structure ◽  
Base Excision

Abstract Oxidative DNA lesions, constantly generated by both endogenous and environmentally induced reactive oxygen species, are removed via the base excision repair pathway. In bacteria, Fpg and Nei DNA glycosylases, belonging to the helix–two-turn–helix (H2TH) structural superfamily, remove oxidised purines and pyrimidines, respectively. Interestingly, the human H2TH family glycosylases, NEIL1, NEIL2 and NEIL3, have been reported to prefer oxidative lesions in DNA bubbles or single-stranded DNA. It had been hypothesised that NEIL2 might be involved in the repair of lesions in transcription bubbles; however, bubble-like structures may appear in other cellular contexts such as displacement loops (D-loops) associated with transcription, recombination or telomere maintenance. The activities of bacterial Fpg and Nei on bubble substrates were not addressed. Also, it is not known whether H2TH enzymes process bubbles containing the third DNA or RNA strand, and how the bubble length and position of the lesion within a bubble affect the excision. We have investigated the removal of 8-oxoguanine (8-oxoG) and 5,6-dihydrouracil (DHU) by Escherichia coli Fpg and Nei and human NEIL1 and NEIL2 from single-strand oligonucleotides, perfect duplexes, bubbles with different numbers of unpaired bases (6–30), bubbles containing the lesion in different positions and D-loops with the third strand made of DNA or RNA. Fpg, NEIL1 and NEIL2 efficiently excised lesions located within bubbles, with NEIL1 and NEIL2 being specific for DHU, and Fpg removing both 8-oxoG and DHU. Nei, in contrast, was significantly active only on DHU located in double-stranded DNA. Fpg and NEIL1 also tolerated the presence of the third strand of either DNA or RNA in D-loops if the lesion was in the single-stranded part, and Fpg, Nei and NEIL1 excised lesions from the double-stranded DNA part of D-loops. The presence of an additional unpaired 5′-tail of DNA or RNA did not affect the activity. No significant position preference for lesions in a 12-mer bubble was found. Overall, the activities of Fpg, NEIL1 and NEIL2 on these non-canonical substrates are consistent with the possibility that these enzymes may participate in the repair in structures arising during transcription or homologous recombination.

scholarly journals Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal

2020 ◽  
Vol 48 (16) ◽  
pp. 9082-9097 ◽  
Author(s):  
Emilie Lebraud ◽  
Guillaume Pinna ◽  
Capucine Siberchicot ◽  
Jordane Depagne ◽  
Didier Busso ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Base Excision Repair ◽  
High Throughput ◽  
Excision Repair ◽  
Genomic Stability ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Base Excision ◽  
Open Question

Abstract One of the most abundant DNA lesions induced by oxidative stress is the highly mutagenic 8-oxoguanine (8-oxoG), which is specifically recognized by 8-oxoguanine DNA glycosylase 1 (OGG1) to initiate its repair. How DNA glycosylases find small non-helix-distorting DNA lesions amongst millions of bases packaged in the chromatin-based architecture of the genome remains an open question. Here, we used a high-throughput siRNA screening to identify factors involved in the recognition of 8-oxoG by OGG1. We show that cohesin and mediator subunits are required for re-localization of OGG1 and other base excision repair factors to chromatin upon oxidative stress. The association of OGG1 with euchromatin is necessary for the removal of 8-oxoG. Mediator subunits CDK8 and MED12 bind to chromatin and interact with OGG1 in response to oxidative stress, suggesting they participate in the recruitment of the DNA glycosylase. The oxidative stress-induced association between the cohesin and mediator complexes and OGG1 reveals an unsuspected function of those complexes in the maintenance of genomic stability.

scholarly journals A non-canonical role for the DNA glycosylase NEIL3 in suppressing APE1 endonuclease-mediated ssDNA damage

2020 ◽  
Vol 295 (41) ◽  
pp. 14222-14235 ◽  
Author(s):  
Anh Ha ◽  
Yunfeng Lin ◽  
Shan Yan
Keyword(s):  
Excision Repair ◽  
Dna Glycosylase ◽  
Genome Integrity ◽  
Ap Endonuclease ◽  
C Terminus ◽  
Egg Extracts ◽  
Lyase Activity ◽  
Repair Pathways ◽  
Base Excision ◽  
Ap Lyase

The DNA glycosylase NEIL3 has been implicated in DNA repair pathways including the base excision repair and the interstrand cross-link repair pathways via its DNA glycosylase and/or AP lyase activity, which are considered canonical roles of NEIL3 in genome integrity. Compared with the other DNA glycosylases NEIL1 and NEIL2, Xenopus laevis NEIL3 C terminus has two highly conserved zinc finger motifs containing GRXF residues (designated as Zf-GRF). It has been demonstrated that the minor AP endonuclease APE2 contains only one Zf-GRF motif mediating interaction with single-strand DNA (ssDNA), whereas the major AP endonuclease APE1 does not. It appears that the two NEIL3 Zf-GRF motifs (designated as Zf-GRF repeat) are dispensable for its DNA glycosylase and AP lyase activity; however, the potential function of the NEIL3 Zf-GRF repeat in genome integrity remains unknown. Here, we demonstrate evidence that the NEIL3 Zf-GRF repeat was associated with a higher affinity for shorter ssDNA than one single Zf-GRF motif. Notably, our protein–protein interaction assays show that the NEIL3 Zf-GRF repeat but not one Zf-GRF motif interacted with APE1 but not APE2. We further reveal that APE1 endonuclease activity on ssDNA but not on dsDNA is compromised by a NEIL3 Zf-GRF repeat, whereas one Zf-GRF motif within NEIL3 is not sufficient to prevent such activity of APE1. In addition, COMET assays show that excess NEIL3 Zf-GRF repeat reduces DNA damage in oxidative stress in Xenopus egg extracts. Together, our results suggest a noncanonical role of NEIL3 in genome integrity via its distinct Zf-GRF repeat in suppressing APE1 endonuclease-mediated ssDNA breakage.

scholarly journals In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities

2017 ◽  
Vol 114 (48) ◽  
pp. E10379-E10388 ◽  
Author(s):  
Isaac A. Chaim ◽  
Zachary D. Nagel ◽  
Jennifer J. Jordan ◽  
Patrizia Mazzucato ◽  
Le P. Ngo ◽  
...  
Keyword(s):  
Excision Repair ◽  
Downstream Processing ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Repair Capacity ◽  
Fluorescent Reporter ◽  
Exogenous Dna ◽  
Interindividual Differences

The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, H2O2, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals.

scholarly journals Structure of a DNA glycosylase that unhooks interstrand cross-links

2017 ◽  
Vol 114 (17) ◽  
pp. 4400-4405 ◽  
Author(s):  
Elwood A. Mullins ◽  
Garrett M. Warren ◽  
Noah P. Bradley ◽  
Brandt F. Eichman
Keyword(s):  
Base Excision Repair ◽  
Pathogenic Bacteria ◽  
Mutational Analysis ◽  
Excision Repair ◽  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Docking Model ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Base Excision

DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

scholarly journals Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis

2021 ◽  
Author(s):  
Brittany L Carroll ◽  
Karl E Zahn ◽  
John P Hanley ◽  
Susan S Wallace ◽  
Julie A Dragon ◽  
...  
Keyword(s):  
Dna Binding ◽  
Large Scale ◽  
Excision Repair ◽  
Dna Glycosylase ◽  
Binding Motif ◽  
Dna Glycosylases ◽  
Main Pathway ◽  
Base Excision ◽  
Reduced Activity ◽  
Insight Into

Abstract Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

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.

Base excision repair mediated cascading triple-signal amplification for the sensitive detection of human alkyladenine DNA glycosylase

The Analyst ◽  
2019 ◽  
Vol 144 (9) ◽  
pp. 3064-3071 ◽  
Author(s):  
Huige Zhang ◽  
Lili Wang ◽  
Yi Xie ◽  
Xianwei Zuo ◽  
Hongli Chen ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Significant Role ◽  
Signal Amplification ◽  
Excision Repair ◽  
Dna Lesions ◽  
Sensitive Detection ◽  
Dna Glycosylase ◽  
Alkyladenine Dna Glycosylase ◽  
Base Excision

DNA glycosylase (DG) plays a significant role in repairing DNA lesions, and the dysregulation of DG activity is associated with a variety of human pathologies.

scholarly journals Non-flipping DNA glycosylase AlkD scans DNA without formation of a stable interrogation complex

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Arash Ahmadi ◽  
Katharina Till ◽  
Paul Hoff Backe ◽  
Pernille Blicher ◽  
Robin Diekmann ◽  
...  
Keyword(s):  
Single Molecule ◽  
Excision Repair ◽  
Dna Glycosylase ◽  
Loss Of Function ◽  
Dna Glycosylases ◽  
Single Molecule Tracking ◽  
Vast Number ◽  
Repair Enzymes ◽  
Base Excision ◽  
Positively Charged

AbstractThe multi-step base excision repair (BER) pathway is initiated by a set of enzymes, known as DNA glycosylases, able to scan DNA and detect modified bases among a vast number of normal bases. While DNA glycosylases in the BER pathway generally bend the DNA and flip damaged bases into lesion specific pockets, the HEAT-like repeat DNA glycosylase AlkD detects and excises bases without sequestering the base from the DNA helix. We show by single-molecule tracking experiments that AlkD scans DNA without forming a stable interrogation complex. This contrasts with previously studied repair enzymes that need to flip bases into lesion-recognition pockets and form stable interrogation complexes. Moreover, we show by design of a loss-of-function mutant that the bimodality in scanning observed for the structural homologue AlkF is due to a key structural differentiator between AlkD and AlkF; a positively charged β-hairpin able to protrude into the major groove of DNA.

Sign in / Sign up

Export Citation Format

Share Document