Temporal coordination of the transcription factor response to H2O2 stress.

Oxidative stress from excess H2O2 activates transcription factors that restore redox balance and repair oxidative damage. Although many transcription factors are activated by H2O2, it is unclear whether they are activated at the same H2O2 concentration, or time. Dose-dependent activation is likely as oxidative stress is not a singular state and exhibits dose-dependent outcomes including cell-cycle arrest and cell death. Here, we show that transcription factor activation is both dose-dependent and coordinated over time. Low levels of H2O2 activate p53, NRF2 and JUN. Yet under high H2O2, these transcription factors are repressed, and FOXO1, NF-κB, and NFAT1 are activated. Time-lapse imaging revealed that the order in which these two groups of transcription factors are activated depends on whether H2O2 is administered acutely by bolus addition, or continuously through the glucose oxidase enzyme. Finally, we provide evidence that 2-Cys peroxiredoxins control which group of transcription factors are activated.

Functional roles and cancer variants of the bifunctional glycosylase NEIL2.

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.

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.

A Human MSH6 Germline Variant Associated With Systemic Lupus Erythematosus Induces Lupus-like Disease in Mice.

OBJECTIVE: To determine if single-nucleotide polymorphisms (SNPs) in DNA repair genes are enriched in individuals with systemic lupus erythematosus (SLE) and if they are sufficient to confer a disease phenotype in a mouse model. METHODS: Human exome chip data of 2499 patients with SLE and 1230 healthy controls were analyzed to determine if variants in 10 different mismatch repair genes (MSH4, EXO1, MSH2, MSH6, MLH1, MSH3, POLH, PMS2, ML3, and APEX2) were enriched in individuals with SLE. A mouse model of the MSH6 SNP, which was found to be enriched in individuals with SLE, was created using CRISPR/Cas9 gene targeting. Wildtype mice and mice heterozygous and homozygous for the MSH6 variant were then monitored for 2 years for the development of autoimmune phenotypes, including the presence of high levels of antinuclear antibodies (ANA). Additionally, somatic hypermutation frequencies and spectra of the intronic region downstream of the VH J558-rearranged JH4 immunoglobulin gene was characterized from Peyer's patches. RESULTS: Based on the human exome chip data, the MSH6 variant (rs63750897, p.Ser503Cys) is enriched among patients with SLE versus controls after we corrected for ancestry (odds ratio = 8.39, P = 0.0398). Mice homozygous for the MSH6 variant (Msh6S502C/S502C ) harbor significantly increased levels of ANA. Additionally, the Msh6S502C/S502C mice display a significant increase in the infiltration of CD68+ cells (a marker for monocytes and macrophages) into the lung alveolar space as well as apoptotic cells. Furthermore, characterization of somatic hypermutation in these mice reveals an increase in the DNA polymerase η mutational signature. CONCLUSION: An MSH6 mutation that is enriched in humans diagnosed with lupus was identified. Mice harboring this Msh6 mutation develop increased autoantibodies and an inflammatory lung disease. These results suggest that the human MSH6 variant is linked to the development of SLE.

The hematopoietic compartment is sufficient for lupus development resulting from the POLB-Y265C mutation.

Systemic lupus erythematosus is a chronic disease characterized by autoantibodies, renal and cutaneous disease, and immune complex formation. Emerging evidence suggests that aberrant DNA repair is an underlying mechanism of lupus development. We previously showed that the POLBY265C/C mutation, which results in development of an aberrant immune repertoire, leads to lupus-like disease in mice. To address whether the hematopoietic compartment is sufficient for lupus development, we transplanted bone marrow cells from POLBY265C/C and POLB+/+ into wild-type congenic mice. Only mice transplanted with the POLBY265C/C bone marrow develop high levels of antinuclear antibodies and renal disease. In conclusion, we show that the hematopoietic compartment harvested from the POLBY265C/C mice is sufficient for development of autoimmune disease.

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.

DNA Polymerase ß in the Context of Cancer.

DNA polymerase beta (Pol ß) is a 39 kD vertebrate polymerase that lacks proofreading ability, yet still maintains a moderate fidelity of DNA synthesis. Pol ß is a key enzyme that functions in the base excision repair and non-homologous end joining pathways of DNA repair. Mechanisms of fidelity for Pol ß are still being elucidated but are likely to involve dynamic conformational motions of the enzyme upon its binding to DNA and deoxynucleoside triphosphates. Recent studies have linked germline and somatic variants of Pol ß with cancer and autoimmunity. These variants induce genomic instability by a number of mechanisms, including error-prone DNA synthesis and accumulation of single nucleotide gaps that lead to replication stress. Here, we review the structure and function of Pol ß, and we provide insights into how structural changes in Pol ß variants may contribute to genomic instability, mutagenesis, disease, cancer development, and impacts on treatment outcomes.

The role of cysteines in the structure and function of OGG1.

8-Oxoguanine glycosylase (OGG1) is a base excision repair enzyme responsible for the recognition and removal of 8-oxoguanine, a commonly occurring oxidized DNA modification. OGG1 prevents the accumulation of mutations and regulates the transcription of various oxidative stress-response genes. In addition to targeting DNA, oxidative stress can affect proteins like OGG1 itself, specifically at cysteine residues. Previous work has shown that the function of OGG1 is sensitive to oxidants, with the cysteine residues of OGG1 being the most likely site of oxidation. Due to the integral role of OGG1 in maintaining cellular homeostasis under oxidative stress, it is important to understand the effect of oxidants on OGG1 and the role of cysteines in its structure and function. In this study, we investigate the role of the cysteine residues in the function of OGG1 by mutating and characterizing each cysteine residue. Our results indicate that the cysteines in OGG1 fall into four functional categories: those that are necessary for (1) glycosylase activity (C146 and C255), (2) lyase activity (C140S, C163, C241, and C253), and (3) structural stability (C253) and (4) those with no known function (C28 and C75). These results suggest that under conditions of oxidative stress, cysteine can be targeted for modifications, thus altering the response of OGG1 and affecting its downstream cellular functions.

Differential immunomodulatory effect of PARP inhibition in BRCA1 deficient and competent tumor cells.

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.

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.

Timing Is Everything: Misincorporation of 8oxodG during Mitosis Is Lethal.

Exploiting universal cancer vulnerabilities has been used as an approach for developing targeted therapies. In this issue of Cancer Research, Rudd and colleagues show that the dual-functioning inhibitor TH588 potentiates the accumulation of reactive oxygen species during mitosis in cancer by disturbing mitotic progression and simultaneously inhibiting the hydrolysis of 8oxodGTP. This leads to increased incorporation of 8oxodG into the DNA during mitotic replication and increased toxicity. Understanding the mechanism of this inhibitor lays the groundwork for identifying cancer targets.See related article by Rudd et al., p. 3530.

Expression of a germline variant in the N-terminal domain of the human DNA glycosylase NTHL1 induces cellular transformation without impairing enzymatic function or substrate specificity.

Oxidatively-induced DNA damage, widely accepted as a key player in the onset of cancer, is predominantly repaired by base excision repair (BER). BER is initiated by DNA glycosylases, which locate and remove damaged bases from DNA. NTHL1 is a bifunctional DNA glycosylase in mammalian cells that predominantly removes oxidized pyrimidines. In this study, we investigated a germline variant in the N-terminal domain of NTHL1, R33K. Expression of NTHL1 R33K in human MCF10A cells resulted in increased proliferation and anchorage-independent growth compared to NTHL1 WT-expressing cells. However, wt-NTHL1 and R33K-NTHL1 exhibited similar substrate specificity, excision kinetics, and enzyme turnover in vitro and in vivo. The results of this study indicate an important function of R33 in BER that is disrupted by the R33K mutation. Furthermore, the cellular transformation induced by R33K-NTHL1 expression suggests that humans harboring this germline variant may be at increased risk for cancer incidence.

Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase ß-DNA complexes.

Eukaryotic DNA polymerase ß (Pol ß) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol ß has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol ß. First, using a doubly labeled DNA construct, we show that Pol ß bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol ß and donor-labeled DNA, we visualized dynamic fingers closing in single Pol ß-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol ß, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol ß reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

DNA polymerase κ: Friend or foe?

In this issue of Science Signaling, Temprine et al report that up-regulation of the translesion DNA polymerase Polκ mediates resistance to BRAF pathway-targeted inhibitors and starvation in melanoma cells. These results exemplify the role that Polκ plays in cellular adaptation to stress.

Revealing an Internal Stabilization Deficiency in the DNA Polymerase ß K289M Cancer Variant through the Combined Use of Chemical Biology and X-ray Crystallography.

The human DNA polymerase (pol) ß cancer variant K289M has altered polymerase activity in vitro, and the structure of wild-type pol ß reveals that the K289 side chain contributes to a network of stabilizing interactions in a C-terminal region of the enzyme distal to the active site. Here, we probed the capacity of the K289M variant to tolerate strain introduced within the C-terminal region and active site. Strain was imposed by making use of a dGTP analogue containing a CF2 group substitution for the ß-γ bridging oxygen atom. The ternary complex structure of the K289M variant displays an alteration in the C-terminal region, whereas the structure of wild-type pol ß is not altered in the presence of the dGTP CF2 analogue. The alteration in the K289M variant impacts the active site, because the enzyme in the ternary complex fails to adopt the normal open to closed conformational change and assembly of the catalytically competent active site. These results reveal the importance of the K289-mediated stabilizing network in the C-terminal region of pol ß and suggest an explanation for why the K289M cancer variant is deficient in polymerase activity even though the position 289 side chain is distal to the active site.

A pre-catalytic non-covalent step governs DNA polymerase ß fidelity.

DNA polymerase ß (pol ß) selects the correct deoxyribonucleoside triphosphate for incorporation into the DNA polymer. Mistakes made by pol ß lead to mutations, some of which occur within specific sequence contexts to generate mutation hotspots. The adenomatous polyposis coli (APC) gene is mutated within specific sequence contexts in colorectal carcinomas but the underlying mechanism is not fully understood. In previous work, we demonstrated that a somatic colon cancer variant of pol ß, K289M, misincorporates deoxynucleotides at significantly increased frequencies over wild-type pol ß within a mutation hotspot that is present several times within the APC gene. Kinetic studies provide evidence that the rate-determining step of pol ß catalysis is phosphodiester bond formation and suggest that substrate selection is governed at this step. Remarkably, we show that, unlike WT, a pre-catalytic step in the K289M pol ß kinetic pathway becomes slower than phosphodiester bond formation with the APC DNA sequence but not with a different DNA substrate. Based on our studies, we propose that pre-catalytic conformational changes are of critical importance for DNA polymerase fidelity within specific DNA sequence contexts.

A new perspective on oxidation of DNA repair proteins and cancer.

Reactive oxygen and nitrogen species (RONS) are formed as byproducts of many endogenous cellular processes, in response to infections, and upon exposure to various environmental factors. An increase in RONS can saturate the antioxidation system and leads to oxidative stress. Consequently, macromolecules are targeted for oxidative modifications, including DNA and protein. The oxidation of DNA, which leads to base modification and formation of abasic sites along with single and double strand breaks, has been extensively investigated. Protein oxidation is often neglected and is only recently being recognized as an important regulatory mechanism of various DNA repair proteins. This is a review of the current state of research on the regulation of DNA repair by protein oxidation with emphasis on the correlation between inflammation and cancer.

DNA polymerase beta participates in DNA End-joining.

DNA double strand breaks (DSBs) are one of the most deleterious lesions and if left unrepaired, they lead to cell death, genomic instability and carcinogenesis. Cells combat DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred to as alternative NHEJ (aNHEJ), which joins ends but results in deletions and insertions. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes are poorly understood. Emerging evidence indicates that X family DNA polymerases lambda (Pol λ) and mu (Pol µ) promote DNA end-joining. Here, we show that DNA polymerase beta (Pol ß), another member of the X family of DNA polymerases, plays a role in aNHEJ. In the absence of DNA Pol ß, fewer small deletions are observed. In addition, depletion of Pol ß results in cellular sensitivity to bleomycin and DNA protein kinase catalytic subunit inhibitors due to defective repair of DSBs. In summary, our results indicate that Pol ß in functions in aNHEJ and provide mechanistic insight into its role in this process.

The NEIL1 G83D germline DNA glycosylase variant induces genomic instability and cellular transformation.

Base excision repair (BER) is a key genome maintenance pathway. The NEIL1 DNA glycosylase recognizes oxidized bases, and likely removes damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene is a rare human germline variant that encodes the NEIL1 G83D protein, which is devoid of DNA glycosylase activity. Here we show that expression of G83D NEIL1 in MCF10A immortalized but non-transformed mammary epithelial cells leads to replication fork stress. Upon treatment with hydrogen peroxide, we observe increased levels of stalled replication forks in cells expressing G83D NEIL1 versus cells expressing the wild-type (WT) protein. Double-strand breaks (DSBs) arise in G83D-expressing cells during the S and G2/M phases of the cell cycle. Interestingly, these breaks result in genomic instability in the form of high levels of chromosomal aberrations and micronuclei. Cells expressing G83D also grow in an anchorage independent manner, suggesting that the genomic instability results in a carcinogenic phenotype. Our results are consistent with the idea that an inability to remove oxidative damage in an efficient manner at the replication fork leads to genomic instability and mutagenesis. We suggest that individuals who harbor the G83D NEIL1 variant face an increased risk for human cancer.

DNA Polymerase ß Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the ß-γ Bridging Group of the Incoming dNTP.

The hydrophobic hinge region of DNA polymerase ß (pol ß) is located between the fingers and palm subdomains. The hydrophobicity of the hinge region is important for maintaining the geometry of the binding pocket and for the selectivity of the enzyme. Various cancer-associated pol ß variants in the hinge region have reduced fidelity resulting from a decreased discrimination at the level of dNTP binding. Specifically, I260M, a prostate cancer-associated variant of pol ß, has been shown to have a reduced discrimination during dNTP binding and also during nucleotidyl transfer. To test whether fidelity of the I260M variant is dependent on leaving group chemistry, we employed a toolkit comprising dNTP bisphosphonate analogues modified at the ß-γ bridging methylene to modulate leaving group (pCXYp mimicking PPi) basicity. Construction of linear free energy relationship plots for the dependence of log(kpol) on leaving group pKa4 revealed that I260M catalyzes dNMP incorporation with a marked negative dependence on leaving group basicity, consistent with a chemical transition state, during both correct and incorrect incorporation. Additionally, we provide evidence that I260M fidelity is altered in the presence of some of the analogues, possibly resulting from a lack of coordination between the fingers and palm subdomains in the presence of the I260M mutation.