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.

Development of an Endonuclease V-Assisted Analytical Method for Sequencing Analysis of Deoxyinosine in DNA.

Deoxyinosine (dI) is a highly mutagenic lesion that preferentially pairs with deoxycytidine during replication, which may induce A to G transition and ultimately contribute to carcinogenesis. Therefore, finding the site of dI modification in DNA is of great value for both basic research and clinical applications. Herein, we developed a novel method to sequence the dI modification site in DNA, which utilizes endonuclease V (EndoV)-dependent deamination repair to specifically label the modification site with biotin-14-dATP that allows the affinity enrichment of dI-bearing DNA for sequencing. We have achieved efficient determination of the location of the modified nucleotide in dI-bearing plasmid DNA with the assistance of EndoV-dependent deamination repair. We have also successfully applied this approach to locate the dI modification sites in the mitochondrial DNA of human cells. Our method should be generally applicable for genome-wide sequencing analysis of dI modifications in living organisms.

Polymorphic variant Asp239Tyr of human DNA glycosylase NTHL1 is inactive for removal of a variety of oxidatively-induced DNA base lesions from genomic DNA.

Base excision repair is the major pathway for the repair of oxidatively-induced DNA damage, with DNA glycosylases removing modified bases in the first step. Human NTHL1 is specific for excision of several pyrimidine- and purine-derived lesions from DNA, with loss of function NTHL1 showing a predisposition to carcinogenesis. A rare single nucleotide polymorphism of the Nthl1 gene leading to the substitution of Asp239 with Tyr within the active site, occurs within global populations. In this work, we overexpressed and purified the variant NTHL1-Asp239Tyr (NTHL1-D239Y) and determined the substrate specificity of this variant relative to wild-type NTHL1 using gas chromatography-tandem mass spectrometry with isotope-dilution, and oxidatively-damaged genomic DNA containing multiple pyrimidine- and purine-derived lesions. Wild-type NTHL1 excised seven DNA base lesions with different efficiencies, whereas NTHL1-D239Y exhibited no glycosylase activity for any of these lesions. We also measured the activities of human glycosylases OGG1 and NEIL1, and E. coli glycosylases Nth and Fpg under identical experimental conditions. Different substrate specificities among these DNA glycosylases were observed. When mixed with NTHL1-D239Y, the activity of NTHL1 was not reduced, indicating no substrate binding competition. These results and the inactivity of the variant D239Y toward the major oxidatively-induced DNA lesions points to the importance of the understanding of this variant's role in carcinogenesis and the potential of individual susceptibility to cancer in individuals carrying this variant.

Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability.

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

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

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 ß-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.

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA.

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3' to inosine in single-strand RNA, at a low reaction temperature of 20-25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.

POLE, POLD1, and NTHL1: the last but not the least hereditary cancer-predisposing genes.

POLE, POLD1, and NTHL1 are involved in DNA replication and have recently been recognized as hereditary cancer-predisposing genes, because their alterations are associated with colorectal cancer and other tumors. POLE/POLD1-associated syndrome shows an autosomal dominant inheritance, whereas NTHL1-associated syndrome follows an autosomal recessive pattern. Although the prevalence of germline monoallelic POLE/POLD1 and biallelic NTHL1 pathogenic variants is low, they determine different phenotypes with a broad tumor spectrum overlapping that of other hereditary conditions like Lynch Syndrome or Familial Adenomatous Polyposis. Endometrial and breast cancers, and probably ovarian and brain tumors are also associated with POLE/POLD1 alterations, while breast cancer and other unusual tumors are correlated with NTHL1 pathogenic variants. POLE-mutated colorectal and endometrial cancers are associated with better prognosis and may show favorable responses to immunotherapy. Since POLE/POLD1-mutated tumors show a high tumor mutational burden producing an increase in neoantigens, the identification of POLE/POLD1 alterations could help select patients suitable for immunotherapy treatment. In this review, we will investigate the role of POLE, POLD1, and NTHL1 genetic variants in cancer predisposition, discussing the potential future therapeutic applications and assessing the utility of performing a routine genetic testing for these genes, in order to implement prevention and surveillance strategies in mutation carriers.

NTHL1 in genomic integrity, aging and cancer.

Efficient DNA repair is essential to maintain genomic integrity. An average of 30,000 base lesions per cell are removed daily by the DNA glycosylases of the base excision repair machinery. With the advent of whole genome sequencing, many germline mutations in these DNA glycosylases have been identified and associated with various diseases, including cancer. In this graphical review, we discuss the function of the NTHL1 DNA glycosylase and how genomic mutations and altered function of this protein contributes to cancer and aging. We highlight its role in a rare tumor syndrome, NTHL1-associated polyposis (NAP), and summarize various other polymorphisms in NTHL1 that can induce early hallmarks of cancer, including genomic instability and cellular transformation.

In Vitro Construction of Large-scale DNA Libraries from Fragments Containing Random Regions using Deoxyinosine-containing Oligonucleotides and Endonuclease V.

Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 1013 species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5' end. Treatment of the PCR products with endonuclease V generates 3' overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.

Förster Resonance Energy Transfer Based Biosensor for Targeting the hNTH1-YB1 Interface as a Potential Anticancer Drug Target.

The Y-box binding protein 1 (YB1) is an established metastatic marker: high expression and nuclear localization of YB1 correlate with tumor aggressiveness, drug resistance, and poor patient survival in various tumors. In the nucleus, YB1 interacts with and regulates the activities of several nuclear proteins, including the DNA glycosylase, human endonuclease III (hNTH1). In the present study, we used Förster resonance energy transfer (FRET) and AlphaLISA technologies to further characterize this interaction and define the minimal regions of hNTH1 and YB1 required for complex formation. This work led us to design an original and cost-effective FRET-based biosensor for the rapid in vitro high-throughput screening for potential inhibitors of the hNTH1-YB1 complex. Two pilot screens were carried out, allowing the selection of several promising compounds exhibiting IC50 values in the low micromolar range. Interestingly, two of these compounds bind to YB1 and sensitize drug-resistant breast tumor cells to the chemotherapeutic agent, cisplatin. Taken together, these findings demonstrate that the hNTH1-YB1 interface is a druggable target for the development of new therapeutic strategies for the treatment of drug-resistant tumors. Moreover, beyond this study, the simple design of our biosensor defines an innovative and efficient strategy for the screening of inhibitors of therapeutically relevant protein-protein interfaces.

Deletion of Endonuclease V suppresses chemically induced hepatocellular carcinoma.

Endonuclease V (EndoV) is a conserved inosine-specific ribonuclease with unknown biological function. Here, we present the first mouse model lacking EndoV, which is viable without visible abnormalities. We show that endogenous murine EndoV cleaves inosine-containing RNA in vitro, nevertheless a series of experiments fails to link an in vivo function to processing of such transcripts. As inosine levels and adenosine-to-inosine editing often are dysregulated in hepatocellular carcinoma (HCC), we chemically induced HCC in mice. All mice developed liver cancer, however, EndoV-/- tumors were significantly fewer and smaller than wild type tumors. Opposed to human HCC, adenosine deaminase mRNA expression and site-specific editing were unaltered in our model. Loss of EndoV did not affect editing levels in liver tumors, however mRNA expression of a selection of cancer related genes were reduced. Inosines are also found in certain tRNAs and tRNAs are cleaved during stress to produce signaling entities. tRNA fragmentation was dysregulated in EndoV-/- livers and apparently, inosine-independent. We speculate that the inosine-ribonuclease activity of EndoV is disabled in vivo, but RNA binding allowed to promote stabilization of transcripts or recruitment of proteins to fine-tune gene expression. The EndoV-/- tumor suppressive phenotype calls for related studies in human HCC.

Evolution of Inosine-Specific Endonuclease V from Bacterial DNase to Eukaryotic RNase.

Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.

Defective repair capacity of variant proteins of the DNA glycosylase NTHL1 for 5-hydroxyuracil, an oxidation product of cytosine.

The NTHL1 gene encodes DNA glycosylase, which is involved in base excision repair, and biallelic mutations of this gene result in NTHL1-associated polyposis (NAP), a hereditary disease characterized by colorectal polyposis and multiple types of carcinomas. However, no proper functional characterization of variant NTHL1 proteins has been done so far. Herein, we report functional evaluation of variant NTHL1 proteins to aid in the accurate diagnosis of NAP. First, we investigated whether it would be appropriate to use 5-hydroxyuracil (5OHU), an oxidation product of cytosine, for the evaluation. In the supF forward mutation assay, 5OHU caused an increase of the mutation frequency in human cells, and the C→T mutation was predominant among the 5OHU-induced mutations. In addition, in DNA cleavage activity assay, 5OHU was excised by NTHL1 as well as four other DNA glycosylases (SMUG1, NEIL1, TDG, and UNG2). When human cells overexpressing the five DNA glycosylases were established, it was found that each of the five DNA glycosylases, including NTHL1, had the ability to suppress 5OHU-induced mutations. Based on the above results, we performed functional evaluation of eight NTHL1 variants using 5OHU-containing DNA substrate or shuttle plasmid. The DNA cleavage activity assay showed that the variants of NTHL1, Q90X, Y130X, R153X, and Q287X, but not R19Q, V179I, V217F, or G286S, showed defective repair activity for 5OHU and two other oxidatively damaged bases. Moreover, the supF forward mutation assay showed that the four truncated-type NTHL1 variants showed a reduced ability to suppress 5OHU-induced mutations in human cells. These results suggest that the NTHL1 variants Q90X, Y130X, R153X, and Q287X, but not R19Q, V179I, V217F, or G286S, were defective in 5OHU repair and the alleles encoding them were considered to be pathogenic for NAP.

Genoprotective Effect of Phyllanthus orbicularis Extract Against UVA, UVB, and Solar Radiation.

One approach to protect the human skin against harmful effects of solar ultraviolet (UV) radiation was to use natural products as photoprotectors. In this work, the extract from specie Phyllanthus orbicularis K was evaluated as a protective agent against the photodamage by UVB, UVA artificial lamps, and environmental sunlight exposure. The plasmid DNA solutions were exposed to radiations using the DNA dosimeter system in the presence of plant extract. The DNA repair enzymes, Escherichia coli Formamidopyrimidine-DNA glycosylase (Fpg) and T4 bacteriophage endonuclease V (T4-endo V), were employed to discriminate oxidized DNA damage and cyclobutane pyrimidine dimers (CPD), respectively. The supercoiled and relaxed forms of DNA were separated through electrophoretic migration in agarose gels. These DNA forms were quantified to determine strand break, representing the types of lesion levels. The results showed that, in the presence of P. orbicularis extract, the CPD and oxidative damage were reduced in irradiated DNA samples. The photoprotective effect of extract was more evident for UVB and sunlight radiation than for UVA. This work documented the UV absorbing properties of P. orbicularis aqueous extract and opened up new vistas in its characterization as protective agent against DNA damage induced by environmental sunlight radiation.

NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress.

Endonuclease III-like protein 1 (NTH1) is a DNA glycosylase required for the repair of oxidized bases, such as thymine glycol, within the base excision repair pathway. We examined regulation of NTH1 protein by the ubiquitin proteasome pathway and identified the E3 ubiquitin ligase tripartite motif 26 (TRIM26) as the major enzyme targeting NTH1 for polyubiquitylation. We demonstrate that TRIM26 catalyzes ubiquitylation of NTH1 predominantly on lysine 67 present within the N terminus of the protein in vitro In addition, the stability of a ubiquitylation-deficient protein mutant of NTH1 (lysine to arginine) at this specific residue was significantly increased in comparison to the wild-type protein when transiently expressed in cultured cells. We also demonstrate that cellular NTH1 protein is induced in response to oxidative stress following hydrogen peroxide treatment of cells and that accumulation of NTH1 on chromatin is exacerbated in the absence of TRIM26 through small interfering RNA (siRNA) depletion. Stabilization of NTH1 following TRIM26 siRNA also causes significant acceleration in the kinetics of DNA damage repair and cellular resistance to oxidative stress, which can be recapitulated by moderate overexpression of NTH1. This demonstrates the importance of TRIM26 in regulating the cellular levels of NTH1, particularly under conditions of oxidative stress.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer.

Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.

Insights into the role of endonuclease V in RNA metabolism in Trypanosoma brucei.

Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3' to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.

Human cells contain a factor that facilitates the DNA glycosylase-mediated excision of oxidized bases from occluded sites in nucleosomes.

Reactive oxygen species generate some 20,000 base lesions per human cell per day. The vast majority of these potentially mutagenic or cytotoxic lesions are subject to base excision repair (BER). Although chromatin remodelers have been shown to enhance the excision of oxidized bases from nucleosomes in vitro, it is not clear that they are recruited to and act at sites of BER in vivo. To test the hypothesis that cells possess factors that enhance BER in chromatin, we assessed the capacity of nuclear extracts from human cells to excise thymine glycol (Tg) lesions from exogenously added, model nucleosomes. The DNA glycosylase NTHL1 in these extracts was able to excise Tg from both naked DNA and sites in nucleosomes that earlier studies had shown to be sterically accessible. However, the same extracts were able to excise lesions from sterically-occluded sites in nucleosomes only after the addition of Mg2+/ATP. Gel mobility shift assays indicated that nucleosomes remain largely intact following the Mg2+/ATP -dependent excision reaction. Size exclusion chromatography indicated that the NTHL1-stimulating activity has a relatively low molecular weight, close to that of NTHL1 and other BER glycosylases; column fractions that contained the very large chromatin remodeling complexes did not exhibit this same stimulatory activity. These results indicate that cells possess a factor(s) that promotes the initiation of BER in chromatin, but differs from most known chromatin remodeling complexes.

Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication.

Base lesions in DNA can stall the replication machinery or induce mutations if bypassed. Consequently, lesions must be repaired before replication or in a post-replicative process to maintain genomic stability. Base excision repair (BER) is the main pathway for repair of base lesions and is known to be associated with DNA replication, but how BER is organized during replication is unclear. Here we coupled the iPOND (isolation of proteins on nascent DNA) technique with targeted mass-spectrometry analysis, which enabled us to detect all proteins required for BER on nascent DNA and to monitor their spatiotemporal orchestration at replication forks. We demonstrate that XRCC1 and other BER/single-strand break repair (SSBR) proteins are enriched in replisomes in unstressed cells, supporting a cellular capacity of post-replicative BER/SSBR. Importantly, we identify for the first time the DNA glycosylases MYH, UNG2, MPG, NTH1, NEIL1, 2 and 3 on nascent DNA. Our findings suggest that a broad spectrum of DNA base lesions are recognized and repaired by BER in a post-replicative process.

Identification of SUMO modification sites in the base excision repair protein, Ntg1.

DNA damaging agents are a constant threat to genomes in both the nucleus and the mitochondria. To combat this threat, a suite of DNA repair pathways cooperate to repair numerous types of DNA damage. If left unrepaired, these damages can result in the accumulation of mutations which can lead to deleterious consequences including cancer and neurodegenerative disorders. The base excision repair (BER) pathway is highly conserved from bacteria to humans and is primarily responsible for the removal and subsequent repair of toxic and mutagenic oxidative DNA lesions. Although the biochemical steps that occur in the BER pathway have been well defined, little is known about how the BER machinery is regulated. The budding yeast, Saccharomyces cerevisiae is a powerful model system to biochemically and genetically dissect BER. BER is initiated by DNA N-glycosylases, such as S. cerevisiae Ntg1. Previous work demonstrates that Ntg1 is post-translationally modified by SUMO in response to oxidative DNA damage suggesting that this modification could modulate the function of Ntg1. In this study, we mapped the specific sites of SUMO modification within Ntg1 and identified the enzymes responsible for sumoylating/desumoylating Ntg1. Using a non-sumoylatable version of Ntg1, ntg1ΔSUMO, we performed an initial assessment of the functional impact of Ntg1 SUMO modification in the cellular response to DNA damage. Finally, we demonstrate that, similar to Ntg1, the human homologue of Ntg1, NTHL1, can also be SUMO-modified in response to oxidative stress. Our results suggest that SUMO modification of BER proteins could be a conserved mechanism to coordinate cellular responses to DNA damage.