DNA polymerase β (Pol β) is an important polymerase that functions in DNA repair within the Base Excision Repair and Non-Homologous End-Joining pathways. It is estimated to function in the repair of up to 50,000 DNA lesions per cell per day, within the base excision repair pathway (BER). Given the significant role Pol β plays in repairing DNA, genetic variants of Pol β have the potential to perturb repair, resulting in mutation accumulation which can potentiate cancer formation. Here we identify an unstudied human germline variant of Pol β, the S180R variant (rs1585898410), which introduces a significant amino acid alteration within the dNTP binding pocket of the enzyme. We demonstrate that S180R is a low fidelity variant of Pol β due to its loss of the ability to discriminate correct nucleotides from incorrect nucleotides. We also show that this variant exhibits a much slower rate of nucleotide incorporation, which could further disrupt repair capacity in vivo. Structural data reveal that this variant not only has structural changes that may disrupt dNTP binding but also a loss of primer terminus positioning and dynamic flexibility of the fingers domain in the binary state, which likely are driving the low fidelity of S180R Pol β. This study highlights the importance of binary positioning and nucleotide coordinating residues for maintaining nucleotide selectivity, polymerase function, and fidelity. It also emphasizes the importance of further study of this human germline Pol β variant in vivo.

PURPOSE:Base excision repair (BER) is the predominant pathway for repairing non‑bulky oxidized and alkylated DNA base lesions, and its fidelity depends on the coordinated action of lesion‑specific DNA glycosylases and downstream repair enzymes. This review aims to summarize recent structural, biochemical, and genomic insights into three base excision repair enzymes, MUTYH DNA glycosylase, NTHL1 DNA glycosylase, and DNA polymerase β. CONCLUSION:This review outlines how MUTYH, NTHL1 and DNA polymerase β protect the genome from mutagenesis, highlights major germline variants associated with disease, and synthesizes the current knowledge on the characteristic single base substitution (SBS) mutational signatures that occur when these repair enzymes are dysfunctional.

The Base Excision Repair (BER) pathway involves a highly coordinated series of protein–protein interactions that facilitate the recognition, excision, and repair of damaged bases. Key enzymes such as DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), polynucleotide kinase-phosphatase (PNKP), DNA polymerase b (Pol β), ligase IIIα (LigIIIα), poly (ADP-ribose) polymerases PARP1 and PARP2, and X-ray repair cross-complementing protein 1 (XRCC1) catalyze BER in a tightly regulated molecular network. These interactions ensure the seamless handoff of DNA intermediates between the core enzymes of the BER pathway. Understanding the details of protein–protein interactions in BER provides valuable insights into the molecular underpinnings of DNA repair processes. In this review, we focus on protein–protein interactions between the components of the single-nucleotide BER (SN-BER) pathway and other proteins that interact with BER components and regulate the coordination of the pathway. We also briefly discuss the interactions of other proteins that interact with the components of SN-BER based on functional evidence.

Genomic DNA is continually exposed to endogenous and exogenous stressors, leading to DNA damage and genomic instability. The base excision repair (BER) mechanism is crucial for maintaining genetic integrity by repairing damaged nucleobases. BER begins when DNA glycosylases cleave the N-glycosidic bond to remove oxidized pyrimidines. DNA glycosylases can be monofunctional or bifunctional. In this study, we explore the bifunctional NTHL1 variant in inflammation-associated colorectal cancer (CRC). NTHL1 tumor syndrome is linked to increased lifetime risks of CRC, breast cancer, and colorectal polyposis. The rare polymorphism rs3087468 (D239Y) results in a loss of glycosylase activity and acts as a dominant-negative mutation. We hypothesize that the NTHL1-D239Y variant increases CRC susceptibility under chronic inflammation in the AOM/DSS cancer initiation model. Male and female C57BL/6 mice (6-8 weeks old) with wild-type (NTHL1WT/WT), heterozygous (NTHL1WT/D239Y), or homozygous (NTHL1D239Y/D239Y) NTHL1 variants were used. In the first experiment, mice were injected with 7.5 mg/kg azoxymethane (AOM), followed by three cycles of 2.5% dextran sulfate sodium (DSS) (5 days DSS alternating with 14 days of normal water). The second experiment used DSS alone. We monitored body weight, collected fecal samples for 16S rRNA sequencing, and measured colon length and polyp number/size at euthanasia. Histology was assessed for inflammation and tumor development. Blood samples were analyzed for micronuclei formation, and immunohistochemistry (IHC) staining was used for tissue proliferation. In the AOM/DSS model, male NTHL1WT/D239Y and NTHL1D239Y/D239Y mice had significantly more polyps compared to NTHL1WT/WT controls. Female NTHL1D239Y/D239Y mice had more polyps than NTHL1WT/D239Y females. In the DSS-only experiment, male NTHL1WT/D239Y mice developed a significant number of polyps compared to wild-type males, while female NTHL1D239Y/D239Y mice developed more polyps than NTHL1WT/WT females. No significant differences in polyp size were observed in the DSS-only experiment. Tumors extending into the proximal colon were seen only in female NTHL1D239Y/D239Y mice. Male NTHL1WT/D239Y mice developed high-grade adenomas in the distal colon compared to wild-type males.Micronucleated erythrocytes were significantly higher in male NTHL1D239Y/D239Y mice compared to controls, but this was not observed in the DSS-only group. No changes in colitis score or colon length were observed. Genotype or AOM/DSS treatment did not affect microbial alpha diversity, but treatment-dependent changes in microbial composition were noted based on sex and genotype. IHC data indicated elevated KI67 expression in NTHL1D239Y/D239Y and NTHL1WT/D239Y mice following AOM/DSS treatment. The NTHL1-D239Y variant promotes malignancy only under chronic inflammation and AOM/DSS treatment. This variant confers a complex, sex- and zygosity-dependent susceptibility to inflammation-associated CRC, with females showing increased tumor development in the proximal colon and males displaying enhanced tumor size and progression. Tarek Michael Masannat, Fayez K. Ghishan, Pawel R. Kiela, Joann B. Sweasy. NTHL1-D239Y variant enhances tumorigenesis in colorectal cancer through cancer initiation and chronic inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB304.

DNA polymerase beta (Pol beta) fills single nucleotide gaps during base excision repair. Deficiencies in Pol beta can lead to increased mutagenesis and genomic instability in the cell, resulting in cancer. Our laboratory has previously shown that the I260 M somatic mutation of Pol beta, which was first identified in prostate cancer, has reduced nucleotide discrimination in a sequence context-dependent manner. I260 M incorporates the incorrect G opposite A in this context more readily than WT. To identify the molecular mechanism of the reduced fidelity of I260M, we studied incorporation using single turnover kinetics and the nature and rates of conformational changes using steady-state fluorescence and Forster resonance energy transfer (FRET). Our data indicate that the I260 M mutation affects the fingers region of rat Pol beta by creating a "collapsed" state in both the open (in the absence of nucleotide) and closed (prior to chemistry) states. I260 M is a temperature-sensitive mutator and binds nucleotides tighter than the WT protein, resulting in reduced fidelity compared to the WT. Additionally, we have generated a kinetic model of WT and I260 M using FRET and single turnover data, which demonstrates that I260 M precatalytic conformation changes differ compared to the WT as it is missing a precatalytic noncovalent step. Taken together, these results suggest that the collapsed state of I260 M may decrease its ability for nucleotide discrimination, illustrating the importance of the "fingers closing" conformational change for polymerase fidelity and accurate DNA synthesis.

The catalytic function of DNA polymerase beta (pol beta) fulfills the gap-filling requirement of the base excision DNA repair pathway by incorporating a single nucleotide into a gapped DNA substrate resulting from the removal of damaged DNA bases. Most importantly, pol beta can select the correct nucleotide from a pool of similarly structured nucleotides to incorporate into DNA in order to prevent the accumulation of mutations in the genome. Pol beta is likely to employ various mechanisms for substrate selection. Here, we use dCTP analogues that have been modified at the beta,gamma-bridging group of the triphosphate moiety to monitor the effect of leaving group basicity of the incoming nucleotide on precatalytic conformational changes, which are important for catalysis and selectivity. It has been previously shown that there is a linear free energy relationship between leaving group pK(a) and the chemical transition state. Our results indicate that there is a similar relationship with the rate of a precatalytic conformational change, specifically, the closing of the fingers subdomain of pol beta. In addition, by utilizing analogue beta,gamma-CHX stereoisomers, we identified that the orientation of the beta,gamma-bridging group relative to R183 is important for the rate of fingers closing, which directly influences chemistry.

DNA damage is a fundamental molecular cause of genomic instability. Base excision repair (BER) is one line of defense to minimize the potential mutagenicity and/or toxicity derived from damaged nucleobase lesions. However, BER in the context of chromatin, in which eukaryotic genomic DNA is compacted through a hierarchy of DNA-histone protein interactions, is not fully understood. Here, we investigate the activity of BER enzymes at 27 unique geometric locations in a nucleosome core particle (NCP), which is the minimal unit of packaging in chromatin. The BER enzymes include uracil DNA glycosylase (UDG), AP endonuclease 1 (APE1), DNA polymerase β (Pol β), and DNA ligase IIIα complexed with X-ray repair cross complementing group 1 (LigIIIα/XRCC1). This global analysis of BER reveals that initiation of the repair event by UDG is dictated by the rotational position of the lesion. APE1 has robust activity at locations where repair is initiated whereas the repair event stalls at the Pol β nucleotide incorporation step within the central ∼45 bp of nucleosomal DNA. The final step of the repair, catalyzed by LigIIIα/XRCC1, is achieved only in the entry/exit regions of the NCP when nick sites are transiently exposed by unwrapping from the histones. Kinetic assays further elucidate that the location of the damaged lesion modulates enzymatic activity. Notably, these data indicate that some of the BER enzymes can act at a significant number of locations even in the absence of chromatin remodelers or other cellular factors. These results inform genome wide maps of DNA damage and mutations and contribute to our understanding of mutational hotspots and signatures.

Introduction: Persistent genotoxic stress leads to spontaneous DNA damage, which occurs on the order of 104−105 events per cell per day. The base excision repair is the first responder in the repair mechanisms after DNA damage, with DNA glycosylase being the first step in the repair process by binding to the DNA, cleaving the N-glycosidic bond and removing the oxidized pyrimidine DNA lesions. NTHL1 is a pivotal bifunctional DNA glycosylase enzyme involved in base excision repair mechanism. NTHL1 tumor syndrome is characterized by an increased lifetime risk for colorectal cancer (CRC), breast cancer, and colorectal polyposis. A rare rs3087468 polymorphism (D239Y) exhibits no glycosylase activity and may act as a dominant-negative mutation. Hypothesis: NTHL1-D239Y variant conforms susceptibility to inflammation-associated colorectal cancer. Methods: 6-8 weeks/old male and female C57BL/6 wild-type (WT, NTHL1WT/WT), heterozygous (NTHL1WT/D239Y), or homozygous (NTHL1D239Y/D239Y) knock-in mice were injected with 7.5mg/kg azoxymethane (AOM) followed by three cycles of alternating 2.5% dextran sulfate sodium (DSS, 5 days) and normal drinking water (14 days). We monitored body weight, collected fecal samples for microbial 16S rRNA amplicon sequencing, and analyzed colon length and number/size of polyps at euthanasia. The histology score was assessed by an unbiased pathologist to evaluate inflammation and tumor development. Blood samples were used to evaluate micronuclei formation. Results: NTHL1WT/D239Y and NTHL1D239Y/D239Y mice showed more pronounced weight loss starting from the 2nd DSS cycle. The number and size of polyps in the distal colon of NTHL1WT/D239Y and NTHL1D239Y/D239Y male mice were increased compared to WT littermates. NTHL1D239Y/D239Y females, but not males, showed increased polyp number compared to NTHL1WT/D239Y mice. No statistical difference was observed in the polyps’ size. Tumors extending into the proximal colon were observed uniquely in NTHL1D239Y/D239Y females. On the other hand, only NTHL1WT/D239Y males developed high adenomas in the distal colon compared to WT males. Interestingly, only NTHL1D239Y/D239Y males showed statistical difference in micronucleated normochromatic erythrocytes compared to WT mice. NTHL1 genotype did not significantly affect fecal microbial α- or β-diversity or colitis score and colon length in either gender. Conclusion: Our data suggest that NTHL1-D239Y variant confers a complex, zygosity- and sex-dependent susceptibility risk for inflammation-associated colorectal cancer that may be intrinsic and independent of changes in the gut microbiota. In females, it promotes more proximal tumor location, while in males, it promotes the size and tumor progression. 5P30CA023074 (JBS), Phoenix Women's Board of the Steele Children's Research Center (FKG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oxidative stress from excess H2O2 activates transcription factors (TFs) that restore redox balance and repair oxidative damage. Though many TFs are activated by H2O2, it is unknown whether they are activated at the same H2O2 concentration or time after H2O2 stress. We found TF activation is tightly coordinated over time and dose dependent. We first focused on p53 and FOXO1 and found that in response to low H2O2, p53 is activated rapidly while FOXO1 remains inactive. In contrast, cells respond to high H2O2 in two temporal phases. In the first phase FOXO1 rapidly shuttles to the nucleus while p53 remains inactive. In the second phase FOXO1 shuts off and p53 levels rise. Other TFs are activated in the first phase with FOXO1 (NF-κB, NFAT1), or the second phase with p53 (NRF2, JUN), but not both. The two phases result in large differences in gene expression. Finally, we provide evidence that 2-Cys peroxiredoxins control which TF are activated and the timing of TF activation.

Pioneer of cell mutagenesis and DNA repair research.

R438W Pol lambda induces cellular transformation in the absence of estrogen.

Treatment with estrogen does not affect cellular proliferation.

Representative western of Pol lambda expression in HME1 cells.

Over 70,000 DNA lesions occur in the cell every day, and the inability to properly repair them can lead to mutations and destabilize the genome, resulting in carcinogenesis. The base excision repair (BER) pathway is critical for maintaining genomic integrity by repairing small base lesions, abasic sites and single-stranded breaks. Monofunctional and bifunctional glycosylases initiate the first step of BER by recognizing and excising specific base lesions, followed by DNA end processing, gap filling, and finally nick sealing. The Nei-like 2 (NEIL2) enzyme is a critical bifunctional DNA glycosylase in BER that preferentially excises cytosine oxidation products and abasic sites from single-stranded, double-stranded, and bubble-structured DNA. NEIL2 has been implicated to have important roles in several cellular functions, including genome maintenance, participation in active demethylation, and modulation of the immune response. Several germline and somatic variants of NEIL2 with altered expression and enzymatic activity have been reported in the literature linking them to cancers. In this review, we provide an overview of NEIL2 cellular functions and summarize current findings on NEIL2 variants and their relationship to cancer.

Supporting Information Figure Legends from Estrogen Drives Cellular Transformation and Mutagenesis in Cells Expressing the Breast Cancer–Associated R438W DNA Polymerase Lambda Protein

Flowchart of the identification of SNPs from breast cancer patients.

Over 70,000 DNA lesions occur in the cell every day, and the inability to properly repair them can lead to mutations and destabilize the genome, resulting in carcinogenesis. The base excision repair (BER) pathway is critical for maintaining genomic integrity by repairing small base lesions, abasic sites and single-stranded breaks. Monofunctional and bifunctional glycosylases initiate the first step of BER by recognizing and excising specific base lesions, followed by DNA end processing, gap filling, and finally nick sealing. The Nei-like 2 (NEIL2) enzyme is a critical bifunctional DNA glycosylase in BER that preferentially excises cytosine oxidation products and abasic sites from single-stranded, double-stranded, and bubble-structured DNA. NEIL2 has been implicated to have important roles in several cellular functions, including genome maintenance, participation in active demethylation, and modulation of the immune response. Several germline and somatic variants of NEIL2 with altered expression and enzymatic activity have been reported in the literature linking them to cancers. In this review, we provide an overview of NEIL2 cellular functions and summarize current findings on NEIL2 variants and their relationship to cancer.