Unique Structural Features of Mammalian NEIL2 DNA Glycosylase Prime Its Activity for Diverse DNA Substrates and Environments.
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ABSTRACT:
SUBMITTER: Eckenroth BE
PROVIDER: S-EPMC7796977 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell survival largely depends on the faithful maintenance of genetic material since genomic DNA is constantly exposed to genotoxicants from both endogenous and exogenous sources. The evolutionarily conserved base excision repair (BER) pathway is critical for maintaining genome integrity by eliminating highly abundant and potentially mutagenic oxidized DNA base lesions. BER is a multistep process, which is initiated with recognition and excision of the DNA base lesion by a DNA glycosylase, followed by DNA end processing, gap filling and finally sealing of the nick. Besides genome maintenance by global BER, DNA glycosylases have been found to play additional roles, including preferential repair of oxidized lesions from transcribed genes, modulation of the immune response, participation in active DNA demethylation and maintenance of the mitochondrial genome. Central to these functions is the DNA glycosylase NEIL2. Its loss results in increased accumulation of oxidized base lesions in the transcribed genome, triggers an immune response and causes early neurodevelopmental defects, thus emphasizing the multitasking capabilities of this repair protein. Here we review the specialized functions of NEIL2 and discuss the consequences of its absence both in vitro and in vivo.
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Project description:Cockayne Syndrome is a segmental premature aging syndrome, which can be caused by loss of function of the CSB protein. CSB is essential for genome maintenance and has numerous interaction partners with established roles in different DNA repair pathways including transcription coupled nucleotide excision repair and base excision repair. Here, we describe a new interaction partner for CSB, the DNA glycosylase NEIL2. Using both cell extracts and recombinant proteins, CSB and NEIL2 were found to physically interact independently of DNA. We further found that CSB is able to stimulate NEIL2 glycosylase activity on a 5-hydroxyl uracil lesion in a DNA bubble structure substrate in vitro. A novel 4,6-diamino-5-formamidopyrimidine (FapyA) specific incision activity of NEIL2 was also stimulated by CSB. To further elucidate the biological role of the interaction, immunofluorescence studies were performed, showing an increase in cytoplasmic CSB and NEIL2 co-localization after oxidative stress. Additionally, stalling of the progression of the transcription bubble with ?-amanitin resulted in increased co-localization of CSB and NEIL2. Finally, CSB knockdown resulted in reduced incision of 8-hydroxyguanine in a DNA bubble structure using whole cell extracts. Taken together, our data supports a biological role for CSB and NEIL2 in transcription associated base excision repair.
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Project description:Compromised DNA repair capacity of individuals could play a critical role in the severity of SARS-CoV-2 infection-induced COVID-19. We therefore analyzed the expression of DNA repair genes in publicly available transcriptomic datasets of COVID-19 patients and found that the level of NEIL2, an oxidized base specific mammalian DNA glycosylase, is particularly low in the lungs of COVID-19 patients displaying severe symptoms. Downregulation of pulmonary NEIL2 in CoV-2-permissive animals and postmortem COVID-19 patients validated these results. To investigate the potential roles of NEIL2 in CoV-2 pathogenesis, we infected Neil2-null (Neil2-/-) mice with a mouse-adapted CoV-2 strain and found that Neil2-/- mice suffered more severe viral infection concomitant with increased expression of proinflammatory genes, which resulted in an enhanced mortality rate of 80%, up from 20% for the age matched Neil2+/+ cohorts. We also found that infected animals accumulated a significant amount of damage in their lung DNA. Surprisingly, recombinant NEIL2 delivered into permissive A549-ACE2 cells significantly decreased viral replication. Toward better understanding the mechanistic basis of how NEIL2 plays such a protective role against CoV-2 infection, we determined that NEIL2 specifically binds to the 5'-UTR of SARS-CoV-2 genomic RNA and blocks protein synthesis. Together, our data suggest that NEIL2 plays a previously unidentified role in regulating CoV-2-induced pathogenesis, via inhibiting viral replication and preventing exacerbated proinflammatory responses, and also via its well-established role of repairing host genome damage.
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Project description:Pyridoxal-5'-phosphate (PLP), in addition to its known metabolic functions, inactivates many DNA-dependent enzymes through conjugation to their critical amino groups. We have investigated the ability of PLP to inhibit bifunctional DNA repair glycosylases, which possess a catalytic amine. Of six enzymes tested, only endonuclease VIII-like protein 2 (NEIL2) was significantly inhibited by PLP. The inhibition was due to Schiff base formation between PLP and the enzyme. PLP-conjugated NEIL2 completely lost its ability to bind damaged DNA. Liquid chromatography/nanoelectrospray ionization tandem mass spectrometry of the products of proteolysis of pyridoxylated NEIL2 identified Lys50 as the site of modification. Thus, the beta2/beta3 loop where Lys50 is located in NEIL2 is important for DNA binding, presumably lies next to a phosphate-binding site, and may represent a target for regulation of the enzyme activity.
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Project description:In this report, we have tried to gain molecular insight into a single nucleotide polymorphism (SNP) in the NEIL2 gene previously identified as "cancer risk modifier" for BRCA2 mutation carriers. To that end, we studied the role of this SNP (rs804271) on NEIL2 transcriptional regulation, oxidative DNA damage and genome instability in two independent set of samples: The first one was a series of eighty-six BRCA1 and BRCA2 mutation carriers and eighty non-carrier controls in which we evaluated the effect of the SNP on NEIL2 gene expression and oxidative DNA damage accumulation. The second was a set of twenty lymphoblastoid cell lines (LCLs), thirteen BRCA1 mutation carriers and seven non-carriers control, that were used to analyze the correlation between NEIL2 mRNA and/or protein levels, the oxidative and the double stranded break (DSB) DNA damage levels. Our results suggest that an excessive production of NEIL2 enzyme, associated with the SNP, may have a deleterious effect modifying cancer risk susceptibility in BRCA2 mutation carriers. We hypothesize that due to the SNP impact on NEIL2 transcriptional upregulation, a cascade of events may converge in the accumulation of oxidative DNA damage and its posterior conversion into DSBs for this specific group of patients.
| S-EPMC5777719 | biostudies-literature
Project description:Reactive oxygen species (ROS) generated endogenously or from exogenous sources produce mutagenic DNA lesions. If not repaired, these lesions could lead to genomic instability and, potentially, to cancer development. NEIL2 (EC 4.2.99.18), a mammalian base excision repair (BER) protein and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Perturbation in NEIL2 expression may, therefore, significantly impact BER capacity and promote genomic instability. To characterize the genetic and environmental factors regulating NEIL2 gene expression, we mapped the human NEIL2 transcriptional start site and partially characterized the promoter region of the gene using a luciferase reporter assay. We identified a strong positive regulatory region from nucleotide -206 to +90 and found that expression from this region was contingent on its being isolated from an adjacent strong negative regulatory region located downstream (+49 to +710 bp), suggesting that NEIL2 transcription is influenced by both these regions. We also found that oxidative stress, induced by glucose oxidase treatment, reduced the positive regulatory region expression levels, suggesting that ROS may play a significant role in regulating NEIL2 transcription. In an initial attempt to characterize the underlying mechanisms, we used in silico analysis to identify putative cis-acting binding sites for ROS-responsive transcription factors within this region and then used site-directed mutagenesis to investigate their role. A single-base change in the region encompassing nucleotides -206 to +90 abolished the effect of oxidative stress that was observed in the absence of the mutation. Our study is the first to provide an initial partial characterization of the NEIL2 promoter and opens the door for future research aimed at understanding the role of genetic and environmental factors in regulating NEIL2 expression.
| S-EPMC2825343 | biostudies-literature
Project description:The DNA glycosylase NEIL2 plays a central role in maintaining genome integrity, in particular during oxidative stress, by recognizing oxidized base lesions and initiating repair of these via the base excision repair (BER) pathway. Post-translational modifications are important molecular switches that regulate and coordinate the BER pathway, and thereby enable a rapid and fine-tuned response to DNA damage. Here, we report for the first time that human NEIL2 is regulated by phosphorylation. We demonstrate that NEIL2 is phosphorylated by the two kinases cyclin-dependent kinase 5 (CDK5) and protein kinase C (PKC) in vitro and in human SH-SY5Y neuroblastoma cells. The phosphorylation of NEIL2 by PKC causes a substantial reduction in NEIL2 repair activity, while CDK5 does not directly alter the enzymatic activity of NEIL2 in vitro, suggesting distinct modes of regulating NEIL2 function by the two kinases. Interestingly, we show a rapid dephosphorylation of NEIL2 in response to oxidative stress in SH-SY5Y cells. This points to phosphorylation as an important modulator of NEIL2 function in this cellular model, not least during oxidative stress.
| S-EPMC9952225 | biostudies-literature
Project description:Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6-7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients' genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Pol?, Lig III? and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Pol?. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.
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Project description:Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes.
| S-EPMC4787834 | biostudies-literature
Project description:The effects of abnormalities in the DNA glycosylases NEIL1, NEIL2, and NEIL3 on human cancer have not been fully elucidated. In this paper, we found that the median somatic total mutation loads and the median somatic single nucleotide mutation loads exhibited significant inverse correlations with the median NEIL1 and NEIL2 expression levels and a significant positive correlation with the median NEIL3 expression level using data for 13 cancer types from the Cancer Genome Atlas (TCGA) database. A subset of the cancer types exhibited reduced NEIL1 and NEIL2 expressions and elevated NEIL3 expression, and such abnormal expressions of NEIL1, NEIL2, and NEIL3 were also significantly associated with the mutation loads in cancer. As a mechanism underlying the reduced expression of NEIL1 in cancer, the epigenetic silencing of NEIL1 through promoter hypermethylation was found. Finally, we investigated the reason why an elevated NEIL3 expression level was associated with an increased number of somatic mutations in cancer and found that NEIL3 expression was positively correlated with the expression of APOBEC3B, a potent inducer of mutations, in diverse cancers. These results suggested that the abnormal expressions of NEIL1, NEIL2, and NEIL3 are involved in cancer through their association with the somatic mutation load.
| S-EPMC4794593 | biostudies-literature