Unique structural features of mammalian NEIL2 DNA glycosylase prime its activity for diverse DNA substrates and environments

SUMMARYOxidative damage on DNA arising from both endogenous and exogenous sources can result in base modifications that promote errors in replication as well as generate sites of base loss (abasic sites) that present unique challenges to maintaining genomic integrity. These lesions are excised by DNA glycosylases in the first step of the base excision repair pathway. Here we present the first crystal structure of a NEIL2 glycosylase, an enzyme active on cytosine oxidation products and abasic sites. The structure reveals an unusual “open” conformation not seen in NEIL1 or NEIL3 orthologs. NEIL2 is predicted to adopt a “closed” conformation when bound to its substrate. Combined crystallographic and solution scattering studies show the enzyme to be conformationally dynamic in a manner distinct among the NEIL glycosylases and provide insight into the unique substrate preference of the enzyme. Additionally, we characterized three cancer variants of human NEIL2, namely S140N, G230W, and G303R.

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Caught in Motion: Human NTHL1 Undergoes Interdomain Rearrangement Necessary for Catalysis

10.1101/2021.08.13.456075 ◽

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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Effect of DNA Glycosylases OGG1 and Neil1 on Oxidized G-Rich Motif in the KRAS Promoter

International Journal of Molecular Sciences ◽

10.3390/ijms22031137 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1137

Author(s):

Annalisa Ferino ◽

Luigi E. Xodo

Keyword(s):

Oxidative Stress ◽

Base Excision Repair ◽

Excision Repair ◽

Start Site ◽

Dna Glycosylases ◽

Repair Pathway ◽

Transcription Start ◽

G Quadruplex ◽

Base Excision ◽

Regulatory Functions

The promoter of the Kirsten ras (KRAS) proto-oncogene contains, upstream of the transcription start site, a quadruplex-forming motif called 32R with regulatory functions. As guanine under oxidative stress can be oxidized to 8-oxoguanine (8OG), we investigated the capacity of glycosylases 8-oxoguanine glycosylase (OGG1) and endonuclease VIII-like 1 (Neil1) to excise 8OG from 32R, either in duplex or G-quadruplex (G4) conformation. We found that OGG1 efficiently excised 8OG from oxidized 32R in duplex but not in G4 conformation. By contrast, glycosylase Neil1 showed more activity on the G4 than the duplex conformation. We also found that the excising activity of Neil1 on folded 32R depended on G4 topology. Our data suggest that Neil1, besides being involved in base excision repair pathway (BER), could play a role on KRAS transcription.

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

10.1101/2021.09.23.461474 ◽

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.

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Evidence for the Involvement of Nucleotide Excision Repair in the Removal of Abasic Sites in Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.10.3522-3528.2000 ◽

2000 ◽

Vol 20 (10) ◽

pp. 3522-3528 ◽

Cited By ~ 56

Author(s):

Carlos A. Torres-Ramos ◽

Robert E. Johnson ◽

Louise Prakash ◽

Satya Prakash

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Repair Process ◽

Dna Glycosylases ◽

Abasic Sites ◽

Skin Cancers ◽

Nucleotide Excision ◽

Progressive Neurodegeneration ◽

Ap Sites ◽

Base Excision

ABSTRACT In eukaryotes, DNA damage induced by ultraviolet light and other agents which distort the helix is removed by nucleotide excision repair (NER) in a fragment ∼25 to 30 nucleotides long. In humans, a deficiency in NER causes xeroderma pigmentosum (XP), characterized by extreme sensitivity to sunlight and a high incidence of skin cancers. Abasic (AP) sites are formed in DNA as a result of spontaneous base loss and from the action of DNA glycosylases involved in base excision repair. In Saccharomyces cerevisiae, AP sites are removed via the action of two class II AP endonucleases, Apn1 and Apn2. Here, we provide evidence for the involvement of NER in the removal of AP sites and show that NER competes with Apn1 and Apn2 in this repair process. Inactivation of NER in the apn1Δ orapn1Δ apn2Δ strain enhances sensitivity to the monofunctional alkylating agent methyl methanesulfonate and leads to further impairment in the cellular ability to remove AP sites. A deficiency in the repair of AP sites may contribute to the internal cancers and progressive neurodegeneration that occur in XP patients.

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Mechanistic Insight into DNA Damage and Repair in Ischemic Stroke: Exploiting the Base Excision Repair Pathway as a Model of Neuroprotection

Antioxidants and Redox Signaling ◽

10.1089/ars.2010.3451 ◽

2011 ◽

Vol 14 (10) ◽

pp. 1905-1918 ◽

Cited By ~ 33

Author(s):

Peiying Li ◽

Xiaoming Hu ◽

Yu Gan ◽

Yanqin Gao ◽

Weimin Liang ◽

...

Keyword(s):

Dna Damage ◽

Ischemic Stroke ◽

Base Excision Repair ◽

Excision Repair ◽

Repair Pathway ◽

Dna Damage And Repair ◽

Base Excision Repair Pathway ◽

Mechanistic Insight ◽

Base Excision ◽

Insight Into

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

Nucleic Acids Research ◽

10.1093/nar/gkab1162 ◽

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.

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

Journal of Bacteriology ◽

10.1128/jb.181.21.6763-6771.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6763-6771 ◽

Cited By ~ 47

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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Inhibitors of DNA Glycosylases as Prospective Drugs

International Journal of Molecular Sciences ◽

10.3390/ijms21093118 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3118 ◽

Cited By ~ 2

Author(s):

Grigory V. Mechetin ◽

Anton V. Endutkin ◽

Evgeniia A. Diatlova ◽

Dmitry O. Zharkov

Keyword(s):

Clinical Trials ◽

Base Excision Repair ◽

Viral Infections ◽

Excision Repair ◽

Biochemical Process ◽

Dna Glycosylases ◽

Repair Pathway ◽

Repair Pathways ◽

Base Excision ◽

Living Organisms

DNA glycosylases are enzymes that initiate the base excision repair pathway, a major biochemical process that protects the genomes of all living organisms from intrinsically and environmentally inflicted damage. Recently, base excision repair inhibition proved to be a viable strategy for the therapy of tumors that have lost alternative repair pathways, such as BRCA-deficient cancers sensitive to poly(ADP-ribose)polymerase inhibition. However, drugs targeting DNA glycosylases are still in development and so far have not advanced to clinical trials. In this review, we cover the attempts to validate DNA glycosylases as suitable targets for inhibition in the pharmacological treatment of cancer, neurodegenerative diseases, chronic inflammation, bacterial and viral infections. We discuss the glycosylase inhibitors described so far and survey the advances in the assays for DNA glycosylase reactions that may be used to screen pharmacological libraries for new active compounds.

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