scholarly journals Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Yuichiro Shimizu ◽  
Yasuhiro Uchimura ◽  
Naoshi Dohmae ◽  
Hisato Saitoh ◽  
Fumio Hanaoka ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Excision Repair ◽  
Dna Glycosylase ◽  
Abasic Site ◽  
Dna Glycosylases ◽  
Protein Protein Interactions ◽  
E Coli ◽  
Base Excision ◽  
Stimulation Of

We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG) that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity ofE. coliTDG homolog (EcMUG), which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients.

scholarly journals Modulation of the Apurinic/Apyrimidinic Endonuclease Activity of Human APE1 and of Its Natural Polymorphic Variants by Base Excision Repair Proteins

2020 ◽  
Vol 21 (19) ◽  
pp. 7147
Author(s):  
Olga A. Kladova ◽  
Irina V. Alekseeva ◽  
Murat Saparbaev ◽  
Olga S. Fedorova ◽  
Nikita A. Kuznetsov
Keyword(s):  
Dna Repair ◽  
Binding Site ◽  
Base Excision Repair ◽  
Protein Interactions ◽  
Excision Repair ◽  
Dna Glycosylases ◽  
Protein Protein Interactions ◽  
Dna Binding Site ◽  
Polymorphic Variants ◽  
Base Excision

Human apurinic/apyrimidinic endonuclease 1 (APE1) is known to be a critical player of the base excision repair (BER) pathway. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that these proteins interact with APE1 either at upstream or downstream steps of BER. Therefore, we may propose that even a minor disturbance of protein–protein interactions on the DNA template reduces coordination and repair efficiency. Here, the ability of various human DNA repair enzymes (such as DNA glycosylases OGG1, UNG2, and AAG; DNA polymerase Polβ; or accessory proteins XRCC1 and PCNA) to influence the activity of wild-type (WT) APE1 and its seven natural polymorphic variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S) was tested. Förster resonance energy transfer–based kinetic analysis of abasic site cleavage in a model DNA substrate was conducted to detect the effects of interacting proteins on the activity of WT APE1 and its single-nucleotide polymorphism (SNP) variants. The results revealed that WT APE1 activity was stimulated by almost all tested DNA repair proteins. For the SNP variants, the matters were more complicated. Analysis of two SNP variants, R237A and G241R, suggested that a positive charge in this area of the APE1 surface impairs the protein–protein interactions. In contrast, variant R221C (where the affected residue is located near the DNA-binding site) showed permanently lower activation relative to WT APE1, whereas neighboring SNP N222H did not cause a noticeable difference as compared to WT APE1. Buried substitution P311S had an inconsistent effect, whereas each substitution at the DNA-binding site, M270T and R274Q, resulted in the lowest stimulation by BER proteins. Protein–protein molecular docking was performed between repair proteins to identify amino acid residues involved in their interactions. The data uncovered differences in the effects of BER proteins on APE1, indicating an important role of protein–protein interactions in the coordination of the repair pathway.

scholarly journals Molecular Mechanisms Regulating the DNA Repair Protein APE1: A Focus on Its Flexible N-Terminal Tail Domain

2021 ◽  
Vol 22 (12) ◽  
pp. 6308
Author(s):  
David J. López ◽  
José A. Rodríguez ◽  
Sonia Bañuelos
Keyword(s):  
Dna Repair ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Excision Repair ◽  
Tail Domain ◽  
Protein Protein Interactions ◽  
Key Enzyme ◽  
Dna Repair Protein ◽  
Base Excision

APE1 (DNA (apurinic/apyrimidinic site) endonuclease 1) is a key enzyme of one of the major DNA repair routes, the BER (base excision repair) pathway. APE1 fulfils additional functions, acting as a redox regulator of transcription factors and taking part in RNA metabolism. The mechanisms regulating APE1 are still being deciphered. Structurally, human APE1 consists of a well-characterized globular catalytic domain responsible for its endonuclease activity, preceded by a conformationally flexible N-terminal extension, acquired along evolution. This N-terminal tail appears to play a prominent role in the modulation of APE1 and probably in BER coordination. Thus, it is primarily involved in mediating APE1 localization, post-translational modifications, and protein–protein interactions, with all three factors jointly contributing to regulate the enzyme. In this review, recent insights on the regulatory role of the N-terminal region in several aspects of APE1 function are covered. In particular, interaction of this region with nucleophosmin (NPM1) might modulate certain APE1 activities, representing a paradigmatic example of the interconnection between various regulatory factors.

scholarly journals Molecular mechanisms associated with clustered lesion-induced impairment of 8-oxoG recognition by the human glycosylase OGG1

2021 ◽  
Author(s):  
Tao Jiang ◽  
Antonio MONARI ◽  
Elise Dumont ◽  
Emmanuelle Bignon
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Excision Repair ◽  
Structural Features ◽  
Repair Pathway ◽  
Dna Lesion ◽  
Damage Response ◽  
Base Excision ◽  
Structural Signature ◽  
Dynamics Simulations

The 8-oxo-7,8-dihydroguanine, referred to as 8-oxoG, is a highly mutagenic DNA lesion that can provoke the appearance of mismatches if it escapes the DNA Damage Response. The specific recognition of its structural signature by the hOGG1 glycosylase is the first step along the Base Excision Repair pathway, that ensures the integrity of the genome by preventing the emergence of mutations. 8-oxoG formation, structural features and repair have been the matter of extensive research and more recently this active field of research expended to the more complicated case of 8-oxoG within clustered lesions. Indeed, the presence of a second lesion within 1 or 2 helix turns can dramatically impact the repair yields of 8-oxoG by glycosylases. In this work, we use mu-range molecular dynamics simulations and machine learning-based post-analysis to explore the molecular mechanisms associated with the recognition of 8-oxoG by hOGG1 when embedded in a multiple lesions site with a mismatch in 5' or 3'. We delineate the stiffening of the DNA-protein interactions upon the presence of the mismatches, and rationalize the much lower repair yields reported with a 5' mismatch by describing the perturbation of 8-oxoG structural features upon addition of an adjacent lesion.

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 Helicobacter pylori Genes Involved in Avoidance of Mutations Induced by 8-Oxoguanine

2006 ◽  
Vol 188 (21) ◽  
pp. 7464-7469 ◽  
Author(s):  
Aurélie Mathieu ◽  
Eyleen J. O'Rourke ◽  
J. Pablo Radicella
Keyword(s):  
Helicobacter Pylori ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Chromosomal Rearrangements ◽  
Dna Glycosylase ◽  
Endonuclease Activity ◽  
Abasic Site ◽  
E Coli ◽  
Cell Extracts ◽  
H Pylori

ABSTRACT Chromosomal rearrangements and base substitutions contribute to the large intraspecies genetic diversity of Helicobacter pylori. Here we explored the base excision repair pathway for the highly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), a ubiquitous form of oxidized guanine. In most organisms, 8-oxoG is removed by a specific DNA glycosylase (Fpg in bacteria or OGG1 in eukaryotes). In the case where replication of the lesion yields an A/8-oxoG base pair, a second DNA glycosylase (MutY) can excise the adenine and thus avoid the fixation of the mutation in the next round of replication. In a genetic screen for H. pylori genes complementing the hypermutator phenotype of an Escherichia coli fpg mutY strain, open reading frame HP0142, a putative MutY coding gene, was isolated. Besides its capacity to complement E. coli mutY strains, HP0142 expression resulted in a strong adenine DNA glycosylase activity in E. coli mutY extracts. Consistently, the purified protein also exhibited such an activity. Inactivation of HP0142 in H. pylori resulted in an increase in spontaneous mutation frequencies. An Mg-dependent AP (abasic site) endonuclease activity, potentially allowing the processing of the abasic site resulting from H. pylori MutY activity, was detected in H. pylori cell extracts. Disruption of HP1526, a putative xth homolog, confirmed that this gene is responsible for the AP endonuclease activity. The lack of evidence for an Fpg/OGG1 functional homolog is also discussed.

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 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.

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