Neil 1 deficiency facilitates chemoresistance through upregulation of RAD18 expression in ovarian cancer stem cells.

Over the past decades, cancer stem cells (CSCs) have emerged as a critical subset of tumor cells associated with tumor recurrence and resistance to chemotherapy. Understanding the mechanisms underlying CSC-mediated chemoresistance is imperative for improving cancer therapy outcomes. This study delves into the regulatory role of NEIL1, a DNA glycosylase, in chemoresistance in ovarian CSCs. We first observed a decreased expression of NEIL1 in ovarian CSCs, suggesting its potential involvement in CSC regulation. Using pan-cancer analysis, we confirmed the diminished NEIL1 expression in ovarian tumors compared to normal tissues. Furthermore, NEIL1 downregulation correlated with an increase in stemness markers and enrichment of CSCs, highlighting its role in modulating CSC phenotype. Further mechanistic investigation revealed an inverse correlation between NEIL1 and RAD18 expression in ovarian CSCs. NEIL1 depletion led to heightened RAD18 expression, promoting chemoresistance possibly via enhancing Translesion DNA Synthesis (TLS)-mediated DNA lesion bypass. Moreover, dowregulation of NEIL1 results in reduced DNA damage accumulation and suppressed apoptosis in ovarian cancer. Overall, our findings unveil a novel mechanism involving NEIL1 and RAD18 in regulating chemoresistance in ovarian CSCs. Targeting this NEIL1-RAD18 axis may offer promising therapeutic strategies for combating chemoresistance and improving ovarian cancer treatment outcomes.

Endonuclease VIII-like 1 deficiency potentiates nigrostriatal dopaminergic neuron degeneration in a male mouse model of Parkinson's disease.

Parkinson's disease (PD) is a common movement disorder caused by a characteristic loss of dopaminergic neurons in the substantia nigra and degeneration of dopamine terminals in the dorsal striatum. Previous studies have suggested that oxidative stress-induced DNA damage may be involved in PD pathogenesis, as steady-state levels of several types of oxidized nucleobases were shown to be elevated in PD brain tissues. These DNA lesions are normally removed from the genome by base excision repair, which is initiated by DNA glycosylase enzymes such as endonuclease VIII-like 1 (Neil1). In this study, we show that Neil1 plays an important role in limiting oxidative stress-induced degeneration of dopaminergic neurons. In particular, Neil1-deficient male mice exhibited enhanced sensitivity to nigrostriatal degeneration after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and Neil1-deficient animals had higher levels of γH2AX-marked DNA damage than wild-type (WT) controls, regardless of treatment status. Moreover, MPTP-treated Neil1-/- male mice had slightly elevated expression of genes related to the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant pathway. Treatment with the Nrf2 activator, monomethyl fumarate, reduced PD-like behaviors and pathology in Neil1-/- male mice, suggesting that Neil1 is an important defense molecule in an oxidative cellular environment. Taken together, these results suggest that Neil1 DNA glycosylase may play an important role in limiting oxidative stress-mediated PD pathogenesis.

Deregulated DNA damage response network in Behcet's disease.

Behcet's disease (BD) is a chronic, relapsing systemic vasculitis of unknown etiology. Since the DNA repair enzyme NEIL1 has been identified as one of the two genetic risk factors for BD by whole exome study, we examined the potential involvement of the DNA damage response (DDR) network in BD. Peripheral blood mononuclear cells from 26 patients and 26 age-/sex-matched healthy controls were studied. Endogenous DNA damage levels were increased in active BD patients compared to controls or patients in remission. In parallel, BD patients had defective nucleotide excision repair capacity. RNA-sequencing revealed reduced expression of NEIL1 that negatively correlated with DNA damage accumulation. On the other hand, expression of genes involved in senescence and senescence-associated secretory phenotype positively correlated with individual endogenous DNA damage levels. We conclude that deregulated DDR contributes to the proinflammatory environment in BD.

Base Excision Repair: Mechanisms and Impact in Biology, Disease, and Medicine.

Base excision repair (BER) corrects forms of oxidative, deamination, alkylation, and abasic single-base damage that appear to have minimal effects on the helix. Since its discovery in 1974, the field has grown in several facets: mechanisms, biology and physiology, understanding deficiencies and human disease, and using BER genes as potential inhibitory targets to develop therapeutics. Within its segregation of short nucleotide (SN-) and long patch (LP-), there are currently six known global mechanisms, with emerging work in transcription- and replication-associated BER. Knockouts (KOs) of BER genes in mouse models showed that single glycosylase knockout had minimal phenotypic impact, but the effects were clearly seen in double knockouts. However, KOs of downstream enzymes showed critical impact on the health and survival of mice. BER gene deficiency contributes to cancer, inflammation, aging, and neurodegenerative disorders. Medicinal targets are being developed for single or combinatorial therapies, but only PARP and APE1 have yet to reach the clinical stage.

TRIM26 Maintains Cell Survival in Response to Oxidative Stress through Regulating DNA Glycosylase Stability.

Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome integrity and in suppressing mutagenesis. 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like protein 1 (NTH1), and the endonuclease VIII-like proteins 1-3 (NEIL1-3) are the key enzymes that initiate repair through the excision of the oxidized base. We have previously identified that the E3 ubiquitin ligase tripartite motif 26 (TRIM26) controls the cellular response to oxidative stress through regulating both NEIL1 and NTH1, although its potential, broader role in BER is unclear. We now show that TRIM26 is a central player in determining the response to different forms of oxidative stress. Using siRNA-mediated knockdowns, we demonstrate that the resistance of cells to X-ray radiation and hydrogen peroxide generated as a consequence of trim26 depletion can be reversed through suppression of selective DNA glycosylases. In particular, a knockdown of neil1 or ogg1 can enhance sensitivity and DNA repair rates in response to X-rays, whereas a knockdown of neil1 or neil3 can produce the same effect in response to hydrogen peroxide. Our study, therefore, highlights the importance of TRIM26 in balancing cellular DNA glycosylase levels required for an efficient BER response.

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

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.

NEIL1 and NEIL2 Are Recruited as Potential Backup for OGG1 upon OGG1 Depletion or Inhibition by TH5487.

DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway.

Nei-like 1 inhibition results in motor dysfunction and promotes inflammation in Parkinson's disease mice model.

Parkinson's disease (PD) is well known as a neurodegenerative disorder with progressive loss of dopaminergic (DA) neurons. Nei-like 1 (NEIL1) is one of four mammalian DNA glycosylases involved in the progression of various diseases, including neuroinflammation. However, it is still unknown if the expression changes of NEIL1 could contribute to PD progression. In the present study, we established mouse model with PD using 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to explore the effects of NEIL1 on PD development. Here, we found that NEIL1 deletion significantly promoted the motor dysfunction in the wild type mice treated with 6-OHDA. Furthermore, DA neuronal loss was further accelerated by NEIL1 deletion in 6-OHDA-injected mice, as evidenced by the significantly reduced expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT). Furthermore, in PD mice induced by MPTP, remarkably reduced expression of NEIL1 was observed in nigra and striatum of mice. A strong positive correlation was detected in the expression of NEIL1 and the survival rate of DA neurons. Also, NEIL1 ablation further elevated the DA neuronal loss in MPTP-treated mice, accompanied with higher glial activation, as evidenced by the obvious up-regulation of glial fibrillary acidic protein (GFAP) and Ionized calcium-Binding Adapter molecule 1 (Iba1). Moreover, MPTP-triggered inflammation was highly aggravated by the loss of NEIL1 through inducing the expression of pro-inflammatory cytokines and chemokines. In contrast, promoting NEIL1 expression effectively reversedPD progression induced by MPTP in mice. Together, these results demonstrated that NEIL1 insufficiency might be a contributing factor for the progression of PD, which therefore could be considered as a novel candidate to develop effective treatments against PD progression.

The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD.

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.

Inhibition of abasic site cleavage in bubble DNA by multifunctional protein YB-1.

Y-box binding protein 1 (YB-1) is widely known to participate in a multiple DNA and RNA processing events in the living cell. YB-1 is also regarded as a putative component of DNA repair. This possibility is supported by relocalization of YB-1 into the nucleus following genotoxic stress. Increased affinity of YB-1 for damaged DNA, especially in its single-stranded form, and its functional interaction with proteins responsible for the initiation of apurinic/apyrimidinic (AP) site repair, namely, AP endonuclease 1 and DNA glycosylase NEIL1, suggest that YB-1 could be involved in the repair of AP sites as a regulatory protein. Here we show that YB-1 has a significant inhibitory effect on the cleavage of AP sites located in single-stranded DNA and in DNA bubble structures. Such interference may be considered as a possible mechanism to prevent single-stranded intermediates of DNA replication, transcription and repair from being converted into highly genotoxic DNA strand breaks, thus allowing the cell to coordinate different DNA processing mechanisms.

Neil3 and NEIL1 DNA glycosylases remove oxidative damages from quadruplex DNA and exhibit preferences for lesions in the telomeric sequence context.

The telomeric DNA of vertebrates consists of d(TTAGGG)n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.

NEIL1 responds and binds to psoralen-induced DNA interstrand crosslinks.

Recent evidence suggests a role for base excision repair (BER) proteins in the response to DNA interstrand crosslinks, which block replication and transcription, and lead to cell death and genetic instability. Employing fluorescently tagged fusion proteins and laser microirradiation coupled with confocal microscopy, we observed that the endonuclease VIII-like DNA glycosylase, NEIL1, accumulates at sites of oxidative DNA damage, as well as trioxsalen (psoralen)-induced DNA interstrand crosslinks, but not to angelicin monoadducts. While recruitment to the oxidative DNA lesions was abrogated by the anti-oxidant N-acetylcysteine, this treatment did not alter the accumulation of NEIL1 at sites of interstrand crosslinks, suggesting distinct recognition mechanisms. Consistent with this conclusion, recruitment of the NEIL1 population variants, G83D, C136R, and E181K, to oxidative DNA damage and psoralen-induced interstrand crosslinks was differentially affected by the mutation. NEIL1 recruitment to psoralen crosslinks was independent of the nucleotide excision repair recognition factor, XPC. Knockdown of NEIL1 in LN428 glioblastoma cells resulted in enhanced recruitment of XPC, a more rapid removal of digoxigenin-tagged psoralen adducts, and decreased cellular sensitivity to trioxsalen plus UVA, implying that NEIL1 and BER may interfere with normal cellular processing of interstrand crosslinks. While exhibiting no enzymatic activity, purified NEIL1 protein bound stably to psoralen interstrand crosslink-containing synthetic oligonucleotide substrates in vitro. Our results indicate that NEIL1 recognizes specifically and distinctly interstrand crosslinks in DNA, and can obstruct the efficient removal of lethal crosslink adducts.

NEIL1 drives the initiation of colorectal cancer through transcriptional regulation of COL17A1.

Deficiency of DNA repair pathways drives the development of colorectal cancer. However, the role of the base excision repair (BER) pathway in colorectal cancer initiation remains unclear. This study shows that Nei-like DNA glycosylase 1 (NEIL1) is highly expressed in colorectal cancer (CRC) tissues and associated with poorer clinical outcomes. Knocking out neil1 in mice markedly suppresses tumorigenesis and enhances infiltration of CD8+ T cells in intestinal tumors. Furthermore, NEIL1 directly forms a complex with SATB2/c-Myc to enhance the transcription of COL17A1 and subsequently promotes the production of immunosuppressive cytokines in CRC cells. A NEIL1 peptide suppresses intestinal tumorigenesis in ApcMin/+ mice, and targeting NEIL1 demonstrates a synergistic suppressive effect on tumor growth when combined with a nuclear factor κB (NF-κB) inhibitor. These results suggest that combined targeting of NEIL1 and NF-κB may represent a promising strategy for CRC therapy.

Artificial targeting of the NEIL1 DNA glycosylase to the mitochondria.

The human NEIL1 DNA glycosylase is one of 11 mammalian glycosylases that initiate base excision repair. While substrate preference, catalytic mechanism, and structural information of NEIL1's ordered residues are available, limited information on its subcellular localization, compounded by relatively low endogenous expression levels, have impeded our understanding of NEIL1. Here, we employed a previously developed computational framework to optimize the mitochondrial localization signal of NEIL1, enabling the visualization of its specific targeting to the mitochondrion via confocal microscopy. While we observed clear mitochondrial localization and increased glycosylase/lyase activity in mitochondrial extracts from low-moderate NEIL1 expression, high NEIL1 mitochondrial expression levels proved harmful, potentially leading to cell death.

Structural basis of nuclear transport for NEIL DNA glycosylases mediated by importin-alpha.

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/ß and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase.

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Šresolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space group P21212 enabled the successful modeling of 96% of residues in the asymmetric unit, with final Rwork and Rfree values of 0.199 and 0.229, respectively.

Isolation of a novel quercetin derivative from Terminalia chebula and RT-PCR-assisted probing to investigate its DNA repair in hepatoma cells.

Background and purpose: DNA damage can lead to carcinogenesis if replication proceeds without proper repair. This study focused on the purification of a novel quercetin derivative present in Terminalia chebula fruit and studied its protective role in hepatoma cells due to H2O2-DNA damage. Experimental approach: The pure compound obtained from the silica gel column was subjected to structural characterization using spectroscopic techniques. MTT assay was employed to select a non-toxic concentration of the isolated compounds on HepG2 and Chang liver cells. The antigenotoxic property of the compound on HepG2 and Chang liver cells was carried out by alkaline comet assay. Analyses of expression levels of mRNA for two DNA repair enzymes, OGG1 and NEIL1, in HepG2 and Chang liver cells, were carried out using the RT-PCR method. Findings/Results: The pure compound obtained from the fraction-5 of diethyl ether extract was identified as a novel quercetin derivative and named 7-(but-2-en-1-yloxy)-2-(4(but-2-en-1-yloxy)-3-hydroxyphenyl)-3- (hexa-2,4-dien-1-yloxy)-6-hydroxy-4H-chromen-4-one. This compound recorded modest toxicity at the highest concentration tested (percentage cell viability at 100 µg/mL was 64.71 ± 0.38 for HepG2 and 45.32 ± 0.07 for Chang liver cells). The compound has demonstrated noteworthy protection against H2O2-induced DNA damage in both cell lines. Analyses of mRNA expression levels for enzymes OGGI and NEIL1 enzymes in HepG2 and Chang liver cells asserted the protective role of the isolated compound against H2O2-induced DNA damage. Conclusion and implication: The protective effect of a novel quercetin derivative isolated from T. chebula in the hepatoma cells is reported here for the first time.

Decoding the Role of NEIL1 Gene in DNA Repair and Lifespan: A Literature Review with Bioinformatics Analysis.

Longevity, the length of an organism's lifespan, is impacted by environmental factors, metabolic processes, and genetic determinants. The base excision repair (BER) pathway is crucial for maintaining genomic integrity by repairing oxidatively modified base lesions. Nei-like DNA Glycosylase 1 (NEIL1), part of the BER pathway, is vital in repairing oxidative bases in G-rich DNA regions, such as telomeres and promoters. Hence, in this comprehensive review, it have undertaken a meticulous investigation of the intricate association between NEIL1 and longevity. The analysis delves into the multifaceted aspects of the NEIL1 gene, its various RNA transcripts, and the diverse protein isoforms. In addition, a combination of bioinformatic analysis is conducted to identify NEIL1 mutations, transcription factors, and epigenetic modifications, as well as its lncRNA/pseudogene/circRNA-miRNA-mRNA regulatory network. The findings suggest that the normal function of NEIL1 is a significant factor in human health and longevity, with defects in NEIL1 potentially leading to various cancers and related syndromes, Alzheimer's disease, obesity, and diabetes.

Bowhead NEIL1: molecular cloning, characterization, and enzymatic properties.

Nei Like DNA Glycosylase 1 (NEIL1) is a DNA glycosylase, which specifically processes oxidative DNA damage by initiating base excision repair. NEIL1 recognizes and removes bases, primarily oxidized pyrimidines, which have been damaged by endogenous oxidation or exogenous mutagenic agents. NEIL1 functions through a combined glycosylase/AP (apurinic/apyrimidinic)-lyase activity, whereby it cleaves the N-glycosylic bond between the DNA backbone and the damaged base via its glycosylase activity and hydrolysis of the DNA backbone through beta-delta elimination due to its AP-lyase activity. In our study we investigated our hypothesis proposing that the cancer resistance of the bowhead whale can be associated with a better DNA repair with NEIL1 being upregulated or more active. Here, we report the molecular cloning and characterization of three transcript variants of bowhead whale NEIL1 of which two were homologous to human transcripts. In addition, a novel NEIL1 transcript variant was found. A differential expression of NEIL mRNA was detected in bowhead eye, liver, kidney, and muscle. The A-to-I editing of NEIL1 mRNA was shown to be conserved in the bowhead and two adenosines in the 242Lys codon were subjected to editing. A mass spectroscopy analysis of liver and eye tissue failed to demonstrate the existence of a NEIL1 isoform originating from RNA editing. Recombinant bowhead and human NEIL1 were expressed in E. coli and assayed for enzymatic activity. Both bowhead and human recombinant NEIL1 catalyzed, with similar efficiency, the removal of a 5-hydroxyuracil lesion in a DNA bubble structure. Hence, these results do not support our hypothesis but do not refute the hypothesis either.

Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9-RAD1-HUS1 (9-1-1) Complex.

Affinity pulldown is a powerful technique to discover novel interaction partners and verify a predicted physical association between two or more proteins. Pulldown assays capture a target protein fused with an affinity tag and analyze the complexed proteins. Here, we detail methods of pulldown assays for two high-affinity peptide fusion tags, Flag tag (DYKDDDDK) and hexahistidine tag (6xHis), to study protein-protein interactions of human NEIL1 glycosylase and the checkpoint protein complex RAD9-RAD1-HUS1 (9-1-1). We uncover unique interactions between 9-1-1 and NEIL1, which suggest a possible inhibitory role of the disordered, phosphorylated C-terminal region of RAD9 in regulating NEIL1 activity in base excision repair through lack of physical association of 9-1-1 and NEIL1.