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.

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 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 A Novel Role for the DNA Repair Enzyme 8-Oxoguanine DNA Glycosylase in Adipogenesis

2021 ◽  
Vol 22 (3) ◽  
pp. 1152
Author(s):  
Sai Santosh Babu Komakula ◽  
Bhavya Blaze ◽  
Hong Ye ◽  
Agnieszka Dobrzyn ◽  
Harini Sampath
Keyword(s):  
Adipocyte Differentiation ◽  
Excision Repair ◽  
Adipogenic Differentiation ◽  
Weight Maintenance ◽  
Transgenic Animals ◽  
Dna Glycosylase ◽  
Antioxidant Defenses ◽  
Dna Glycosylases ◽  
A Cell ◽  
Base Excision

Cells sustain constant oxidative stress from both exogenous and endogenous sources. When unmitigated by antioxidant defenses, reactive oxygen species damage cellular macromolecules, including DNA. Oxidative lesions in both nuclear and mitochondrial DNA are repaired via the base excision repair (BER) pathway, initiated by DNA glycosylases. We have previously demonstrated that the BER glycosylase 8-oxoguanine DNA glycosylase (OGG1) plays a novel role in body weight maintenance and regulation of adiposity. Specifically, mice lacking OGG1 (Ogg1−/−) are prone to increased fat accumulation with age and consumption of hypercaloric diets. Conversely, transgenic animals with mitochondrially-targeted overexpression of OGG1 (Ogg1Tg) are resistant to age- and diet-induced obesity. Given these phenotypes of altered adiposity in the context of OGG1 genotype, we sought to determine if OGG1 plays a cell-intrinsic role in adipocyte maturation and lipid accumulation. Here, we report that preadipocytes from Ogg1−/− mice differentiate more efficiently and accumulate more lipids than those from wild-type animals. Conversely, OGG1 overexpression significantly blunts adipogenic differentiation and lipid accretion in both pre-adipocytes from Ogg1Tg mice, as well as in 3T3-L1 cells with adenovirus-mediated OGG1 overexpression. Mechanistically, changes in adipogenesis are accompanied by significant alterations in cellular PARylation, corresponding with OGG1 genotype. Specifically, deletion of OGG1 reduces protein PARylation, concomitant with increased adipogenic differentiation, while OGG1 overexpression significantly increases PARylation and blunts adipogenesis. Collectively, these data indicate a novel role for OGG1 in modulating adipocyte differentiation and lipid accretion. These findings have important implications to our knowledge of the fundamental process of adipocyte differentiation, as well as to our understanding of lipid-related diseases such as obesity.

scholarly journals Isoforms of Base Excision Repair Enzymes Produced by Alternative Splicing

2019 ◽  
Vol 20 (13) ◽  
pp. 3279 ◽  
Author(s):  
Boldinova ◽  
Khairullin ◽  
Makarova ◽  
Zharkov
Keyword(s):  
Alternative Splicing ◽  
Base Excision Repair ◽  
Intracellular Transport ◽  
Splice Variants ◽  
Excision Repair ◽  
Dna Glycosylases ◽  
Polymerase Beta ◽  
Repair Enzymes ◽  
Alternatively Spliced ◽  
Base Excision

Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport.

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 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 Repair of Hypoxanthine in DNA Revealed by DNA Glycosylases and Endonucleases From Hyperthermophilic Archaea

2021 ◽  
Vol 12 ◽  
Author(s):  
Tan Lin ◽  
Likui Zhang ◽  
Mai Wu ◽  
Donghao Jiang ◽  
Zheng Li ◽  
...  
Keyword(s):  
Excision Repair ◽  
Dna Glycosylase ◽  
Hyperthermophilic Archaea ◽  
Future Research ◽  
Dna Glycosylases ◽  
Uracil Dna Glycosylase ◽  
Alternative Pathways ◽  
Endonuclease V ◽  
Base Excision ◽  
Dna Strand

Since hyperthermophilic Archaea (HA) thrive in high-temperature environments, which accelerate the rates of deamination of base in DNA, their genomic stability is facing a severe challenge. Hypoxanthine (Hx) is one of the common deaminated bases in DNA. Generally, replication of Hx in DNA before repaired causes AT → GC mutation. Biochemical data have demonstrated that 3-methyladenine DNA glycosylase II (AlkA) and Family V uracil DNA glycosylase (UDG) from HA could excise Hx from DNA, thus triggering a base excision repair (BER) process for Hx repair. Besides, three endonucleases have been reported from HA: Endonuclease V (EndoV), Endonuclease Q (EndoQ), and Endonuclease NucS (EndoNucS), capable of cleaving Hx-containing DNA, thereby providing alternative pathways for Hx repair. Both EndoV and EndoQ could cleave one DNA strand with Hx, thus forming a nick and further initiating an alternative excision repair (AER) process for the follow-up repair. By comparison, EndoNucS cleaves both strands of Hx-containing DNA in a restriction endonuclease manner, thus producing a double-stranded break (DSB). This created DSB might be repaired by homologous recombination (HR) or by a combination activity of DNA polymerase (DNA pol), flap endonuclease 1 (FEN1), and DNA ligase (DNA lig). Herein, we reviewed the most recent advances in repair of Hx in DNA triggered by DNA glycosylases and endonucleases from HA, and proposed future research directions.

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 ◽  
Open Conformation ◽  
Main Pathway ◽  
Base Excision ◽  
Insight Into

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 NTLH1 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 lower binding affinity for 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.

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