Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs.

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.

Binding of anticancer drug adriamycin to parallel G-quadruplex DNA [d-(TTAGGGT)]4 comprising human telomeric DNA leads to thermal stabilization: A multiple spectroscopy study.

Adriamycin is known to exert its anti cancer action by inhibiting DNA duplication, RNA transcription and topoisomerase-II enzyme action. Recent findings of its binding to G-quadruplex DNA resulting in telomere dysfunction indicated multiple strategies of its action. The interaction of anticancer drug adriamycin with parallel stranded inter molecular G-quadruplex DNA [d-(TTAGGGT)]4 comprising human telomeric DNA sequence TTAGGG was investigated by absorption, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy to understand mode of their interaction. The adriamycin binds as monomer to G-quadruplex DNA with affinity (Kb1 = 9.8x105 M-1 and Kb2 = 6.7x105 M-1 ) higher than that reported for daunomycin, at two independent sites, mainly in terminal stacking and groove binding modes. The bound complex formed as a result of specific interactions induces thermal stabilization of DNA by 12.5-28.1°C, which is likely to hinder telomere association with telomerase enzyme and contribute significantly to adriamycin-induced apoptosis in cancer cell lines. The findings have therapeutic potential towards drug designing by way of altering substituent groups on anthracyclines to enhance efficacy using additional mechanism of targeting pathway of telomere maintenance by disrupting telomerase association with telomeres.

Epigenomic promoter alterations predict for benefit from immune checkpoint inhibition in metastatic gastric cancer.

BACKGROUND: Utilization of alternative transcription start sites through alterations in epigenetic promoter regions causes reduced expression of immunogenic N-terminal peptides, which may facilitate immune evasion in early gastric cancer. We hypothesized that tumors with high alternate promoter utilization would be resistant to immune checkpoint inhibition in metastatic gastric cancer. PATIENTS AND METHODS: Two cohorts of patients with metastatic gastric cancer treated with immunotherapy were analyzed. The first cohort (N = 24) included patients treated with either nivolumab or pembrolizumab. Alternate promoter utilization was measured using the NanoString® (NanoString Technologies, Seattle, WA, USA) platform on archival tissue samples. The second cohort was a phase II clinical trial of patients uniformly treated with pembrolizumab (N = 37). Fresh tumor biopsies were obtained, and transcriptomic analysis was carried out on RNAseq data. Alternate promoter utilization was correlated to T-cell cytolytic activity, objective response rate and survival. RESULTS: In the first cohort 8 of 24 (33%) tumors were identified to have high alternate promoter utilization (APhigh), and this was used to define the APhigh tertile of the second cohort (13 APhigh of 37). APhigh tumors exhibited decreased markers of T-cell cytolytic activity and lower response rates (8% versus 42%, P = 0.03). Median progression-free survival was lower in the APhigh group (55 versus 180 days, P = 0.0076). In multivariate analysis, alternative promoter utilization was an independent predictor of immunotherapy survival [hazard ratio 0.29, 95% confidence interval 0.099-0.85, P = 0.024). Analyzing tumoral evolution through paired pre-treatment and post-treatment biopsies, we observed consistent shifts in alternative promoter utilization rate associated with clinical response. CONCLUSION: A substantial proportion of metastatic gastric cancers utilize alternate promoters as a mechanism of immune evasion, and these tumors may be resistant to anti-PD1 immune checkpoint inhibition. Alternate promoter utilization is thus a potential mechanism of resistance to immune checkpoint inhibition, and a novel predictive biomarker for immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT#02589496.

Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA.

The kinetics of thymine-thymine cyclobutane pyrimidine dimer (TT-CPD) formation was studied at 23 thymine-thymine base steps in two 247-base pair DNA sequences irradiated at 254 nm. Damage was assayed site-specifically and simultaneously on both the forward and reverse strands by detecting emission from distinguishable fluorescent labels at the 5'-termini of fragments cleaved at CPD sites by T4 pyrimidine dimer glycosylase and separated by gel electrophoresis. The total DNA strand length of nearly 1000 bases made it possible to monitor damage at all 9 tetrads of the type XTTY, where X and Y are non-thymine bases. TT-CPD yields for different tetrads were found to vary by as much as an order of magnitude, but similar yields were observed at all instances of a given tetrad. Kinetic analysis of CPD formation at 23 distinct sites reveals that both the formation and reversal photoreactions depend sensitively on the identity of the nearest-neighbour bases on the 5' and the 3' side of a photoreactive TT base step. The lowest formation and reversal rates occur when two purine bases flank a TT step, while the highest formation and reversal rates are observed for tetrads with at least one flanking C. Overall, the results show that the probabilities of CPD formation and photoreversal depend principally on interactions with nearest-neighbour bases.

The interaction of cisplatin with a human telomeric DNA sequence containing seventeen tandem repeats.

The anti-tumour drug, cisplatin, preferentially forms adducts at G-rich DNA sequences. Telomeres are found at the ends of chromosomes and, in humans, contain the repeated DNA sequence (GGGTTA)(n) that is expected to be targeted by cisplatin. Using a plasmid clone with 17 tandem telomeric repeats, (GGGTTA)(17), the DNA sequence specificity of cisplatin was investigated utilising the linear amplification procedure that pin-pointed the precise sites of cisplatin adduct formation. This procedure used a fluorescently labelled primer and capillary electrophoresis with laser-induced fluorescence detection to determine the DNA sequence specificity of cisplatin. This technique provided a very accurate analysis of cisplatin-DNA adduct formation in a long telomeric repeat DNA sequence. The DNA sequence specificity of cisplatin in a long telomeric tandem repeat has not been previously reported. The results indicated that the 3'-end of the G-rich strand of the telomeric repeat was preferentially damaged by cisplatin and this suggests that the telomeric DNA repeat has an unusual conformation.

The DNA sequence specificity of bleomycin cleavage in telomeric sequences in human cells.

Bleomycin is an antibiotic drug that is widely used in cancer chemotherapy. Telomeres are located at the ends of chromosomes and comprise the tandemly repeated DNA sequence (GGGTTA)( n ) in humans. Since bleomycin cleaves DNA at 5'-GT dinucleotide sequences, telomeres are expected to be a major target for bleomycin cleavage. In this work, we determined the DNA sequence specificity of bleomycin cleavage in telomeric sequences in human cells. This was accomplished using a linear amplification procedure, a fluorescently labelled oligonucleotide primer and capillary gel electrophoresis with laser-induced fluorescence detection. This represents the first occasion that the DNA sequence specificity of bleomycin cleavage in telomeric DNA sequences in human cells has been reported. The bleomycin DNA sequence selectivity was mainly at 5'-GT dinucleotides, with lesser amounts at 5'-GG dinucleotides. The cellular bleomycin telomeric DNA damage was also compared with bleomycin telomeric damage in purified human genomic DNA and was found to be very similar. The implications of these results for the understanding of bleomycin's mechanism of action in human cells are discussed.

Effects of N-Acetylcysteine on the reproductive performance, oxidative stress and RNA sequencing of Nubian goats.

N-acetylcysteine (NAC) has been found to enhance the protective ability of cells to counter balance oxidative stress and inflammation. To investigate the effects of dietary NAC supplementation on the reproductive performance of goats, the reproductive performance and endometrial transcriptome of goats fed with diets with NAC (NAC group) and without NAC supplementation (control group) were compared. Results showed that the goats fed with 0.03% and 0.05% NAC had similar litter size, birth weight, nitric oxide (NO), sex hormones and amino acids levels compared with the goats of the control group. However, feeding with 0.07% NAC supplementation from day 0 to day 30 of gestation remarkably increased the litter size of goats. The goats of the 0.07% NAC group presented increased levels of NO relative to the control group, but their sex hormones and amino acids showed no differences. Comparative transcriptome analysis identified 207 differentially expressed genes (DEGs) in the endometrium between the control and the 0.07% NAC groups. These DEGs included 146 upregulated genes and 61 downregulated genes in the 0.07% NAC group. They were primarily involved in the cellular response to toxic substances, oxidoreductase activity, immune receptor activity, signalling receptor binding, cytokine-cytokine receptor interactions, PI3K-Akt signalling pathway and PPAR signalling pathway. In conclusion, results showed that dietary 0.07% NAC supplementation exerted a beneficial effect on the survival of goat embryos at the early pregnancy stage. Such positive outcome might be due to the increased NO production and affected expression of genes involved in the anti-inflammation pathways of the endometrium.

Anticancer activities of alkylating pyrrole-imidazole polyamides with specific sequence recognition.

In recent years, many diseases including cancer and hereditary and viral diseases have been understood at the DNA sequence level. Direct control of the expression level of a specific gene would provide a promising approach for knowledge-based therapy. N-methylpyrrole and N-methylimidazole polyamides are a new type of small compound that precisely bind to the minor groove of the DNA duplex in a sequence-specific fashion and recruit alkylating agents to the target sequence. We designed and synthesized a series of sequence-specific alkylating Py-Im polyamide conjugates that selectively alkylate predetermined DNA sequences. We have shown that sequence-specific alkylating agents possess gene-silencing activities when they alkylate coding regions of template strands and show promising potency against human cancer cell lines and xenografts possessing human cancer cells. In this study, we focus on recent progress in alkylating Py-Im polyamides with regard to sequence specificity and biological activities, and the future direction of the rational molecular design of genetic switches in the post-genome era is described.

An analysis of enzyme kinetics data for mitochondrial DNA strand termination by nucleoside reverse transcription inhibitors.

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-gamma hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-gamma) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-gamma with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication.

Isolation and characterization of enantioselective DNA aptamers for ibuprofen.

Single stranded DNA aptamers that can bind to ibuprofen, a widely used anti-inflammation drug, were selected from random DNA library of 10(15) nucleotides by FluMag-SELEX process. Five different sequences were selected and their enantioselectivity and affinity were characterized. Three out of five aptamer candidates did not show any affinity to (S)-ibuprofen, but only to racemic form of ibuprofen, suggesting that they are (R)-ibuprofen specific aptamers. Another two aptamer candidates showed affinity to both racemic form and (S)-ibuprofen, which were considered as (S)-ibuprofen specific aptamers. The affinity of five ssDNA aptamers isolated was in a range of 1.5-5.2microM. In addition, all of these five aptamers did not show any affinity to analogues of ibuprofen in its profen's group (fenoprofen, flubiprofen, and naproxen) and the antibiotics of oxytetracycline, another control.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.

The picornavirus avian encephalomyelitis virus possesses a hepatitis C virus-like internal ribosome entry site element.

Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotide-long 5' untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3' end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.

Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): association with aryl hydrocarbon receptor 1 and 2 isoforms.

The present study focuses on the molecular mechanism and interspecies differences in susceptibility of avian aryl hydrocarbon receptor (AHR)-cytochrome P4501A (CYP1A) signaling pathway. By the cloning of 5'-flanking regions of CYP1A5 gene from common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus), seven putative xenobiotic response elements (XREs) were identified within 2.7 kb upstream region of common cormorant CYP1A5 (ccCYP1A5), and six XREs were found within 0.9 kb of chicken CYP1A5 (ckCYP1A5). Analysis of sequential deletion and mutagenesis of the binding sites in avian CYP1A5 genes by in vitro reporter gene assays revealed that two XREs at -613 bp and -1585 bp in ccCYP1A5, and one XRE at -262 bp in ckCYP1A5 conferred TCDD-responsiveness. The binding of AHR1 with AHR nuclear translocator 1 (ARNT1) to the functional XRE in a TCDD-dependent manner was verified with gel shift assays, suggesting that avian CYP1A5 is induced by TCDD through AHR1/ARNT1 signaling pathway as well as mammalian CYP1A1 but through a distinct pathway from mammalian CYP1A2, an ortholog of the CYP1A5. TCDD-EC(50) for the transcriptional activity in both cormorant AHR1- and AHR2-ccCYP1A5 reporter construct was 10-fold higher than that in chicken AHR1-ckCYP1A5 reporter construct. In contrast, chicken AHR2 showed no TCDD-dependent response. The TCDD-EC(50) for CYP1A5 transactivation was altered by switching AHR1 between the two avian species, irrespective of the species from which the regulatory region of CYP1A5 gene originates. Therefore, the structural difference in AHR, not the CYP1A5 regulatory region may be a major factor to account for the dioxin susceptibility in avian species.

5-Bromodeoxyuridine inhibits sequence changes within inverted repeat DNA during embryogenesis.

Previous studies on the genome of Strongylocentrotus purpuratus sea urchin have shown that changes in the nucleotide sequence of inverted repeat sequences occur during embryogenesis. The present study indicates that these sequence changes fail to occur when the embryos are raised in the presence of 5-bromodeoxyuridine. This drug is an analog of thymidine, is incorporated into the DNA during embryogenesis, and inhibits cell differentiation in these embryos.

Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens.

K-region epoxides of the carcinogens benz[a] anthracene, dibenz[a,h]anthracene, and 7-methylbenz[a] anthracene are mutagenic in strains of Salmonella typhimurium designed to detect frameshift mutagens. Parent hydrocarbons, K-region diols and phenols and some other epoxides are inactive as mutagens in these tests. Polycyclic hydrocarbon epoxides, and other presumed proximal carcinogens, are discussed as examples of intercalating agents with reactive side chains. It has been shown previously that intercalating agents with reactive side chains are potent frameshift mutagens.

Omeprazole stimulates the induction of human insulin-like growth factor binding protein-1 through aryl hydrocarbon receptor activation.

5-Methoxy-2-{(4-methoxy-3,5-dimethyl-pyridin-2-yl)methylsulfinyl}-3H-benzoimidazole (omeprazole), a benzoimidazole-derived gastric H(+)/K(+)-ATPase proton pump inhibitor (PPI) extensively prescribed for the treatment of gastroesophageal acid reflux disease, can stimulate the expression of CYP1A1 via activation of the human aryl hydrocarbon receptor (hAhR) in an apparent nonligand-binding manner. Here, we have examined the effect of nonclassical, i.e., nonligand binding, AhR activation by omeprazole upon human insulin-like growth factor binding protein (hIGFBP)-1, a secreted phosphoprotein involved in regulation of insulin-like growth factor-I/II bioavailability and mitogenic activity. Analysis of the proximal promoter of the hIGFBP-1 gene reveals the presence of an aryl hydrocarbon binding/dioxin response element (DRE). Quantitative mRNA analysis revealed hIGFBP-1 expression to be responsive to both ligand (TCDD) and nonligand (omeprazole) modes of hAhR activation in the human hepatocarcinoma HepG2 cell line. Furthermore, mutagenesis of the DRE renders the hIGFBP-1 promoter unresponsive to both compounds in HepG2 cells. Likewise, small interfering RNA-mediated hAhR ablation inhibits TCDD and omeprazole-dependent hIGFBP-1 induction, as determined by quantitative mRNA analysis. Cotreatment with cycloheximide further suggests a direct transcriptional role for hAhR at the hIGFBP-1 promoter. Omeprazole exposure prompted a significant increase in both hIGFBP-1 mRNA and secreted protein from HepG2 cells. In addition, we present in vitro evidence indicating that omeprazole at a concentration comparable with that found circulating in subjects undergoing PPI therapy can stimulate the expression of hIGFBP-1. These data demonstrate that activation of hAhR by pharmaceuticals such as omeprazole can alter IGFBP-1 expression and thus may influence IGFBP-1-dependent physiological processes.

Shiga Toxin as a Potential Trigger of CFHR1 Deletion-Associated Thrombotic Microangiopathy.

Thrombotic microangiopathy (TMA) may result from a variety of clinical conditions, including thrombotic thrombocytopenic purpura, Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome and complement-mediated hemolytic uremic syndrome. Thrombocytopenic purpura is diagnosed when ADAMTS13 is <10%, while a diagnosis of Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome is made with the evidence of infection by Shiga toxin-producing Escherichia coli. Diagnosis of complement-mediated hemolytic uremic syndrome is not dependent on a specific laboratory test and is a diagnosis of exclusion. TMA is a rare disease and finding individuals that have more than 1 concurrent etiology leading to TMA is even more rare. Here we describe the presentation and management of an individual with CFHR1 deletion-associated TMA also found to have a positive stool Shiga toxin. We discuss the significance of Shiga toxin in serving as a trigger for development of TMA in an individual predisposed to development of TMA due to presence of a homozygous deletion in CFHR1.

Dietary genistein supplementation for breeders and their offspring improves the growth performance and immune function of broilers.

Genistein (GEN) is mainly extracted from soy plants and has potential functions as an antioxidant and in promoting immune function and growth. This study evaluated the effects of feeding breeders and their offspring dietary GEN on the immune function and growth performance of broiler chicks. Breeders were assigned to a control diet or GEN diet (control diet +400 mg/kg GEN), and their offspring were fed a control diet or GEN diet (control diet +40 mg/kg GEN). GEN treatment increased the body weight gain, tibial length, tibial width and slaughter performance of broilers and decreased the feed conversion ratio. The treatment also affected skeletal muscle myosin assembly and growth and increased growth hormone levels and IGF-I and IGFBP1 expression. Following GEN treatment, antigen processing and presentation, macrophage activation, B lymphocyte, NK cell and helper T cell proliferation, and CD4+ T lymphocyte differentiation all increased significantly. Increases were also observed in IgM and IgG concentrations, antibody titers, and antioxidant capacity. In addition, GEN treatment activated the Toll-like receptor signaling pathway and MAPK cascade signaling pathway. In summary, dietary GEN supplementation for breeders and their offspring can improve the growth performance and immune function of broiler chicks.

Oxidative DNA base damage in MCF-10A breast epithelial cells at clinically achievable concentrations of doxorubicin.

The cellular metabolism of doxorubicin generates reactive oxygen species with significant potential to damage DNA. Such DNA damage can result in mutations if not adequately repaired by cellular DNA repair pathways. Secondary malignancies have been reported in patients who have received doxorubicin-containing chemotherapeutic regimens; however, the underlying molecular mechanism(s) to explain the development of these tumors remains under active investigation. We have previously demonstrated the presence of DNA bases modified by oxidation in the peripheral blood mononuclear cells of patients with breast cancer following treatment with doxorubicin. In those studies, doxorubicin was administered by continuous infusion over 96 h to minimize the risk of cardiac toxicity. To evaluate potential mechanisms underlying doxorubicin-induced DNA base oxidation in non-malignant tissues, MCF-10A breast epithelial cells were cultured for 96 h with the same doxorubicin concentration achieved in vivo (0.1 microM). During doxorubicin exposure, MCF-10A cells underwent growth arrest and apoptosis, developed elevated levels of reactive oxygen species, and demonstrated a time-dependent and significant increase in the levels of 11 oxidized DNA bases, as determined by gas chromatography/mass spectroscopy. Diminished expression of DNA repair enzymes was also observed over the same time course. Thus, clinically achievable concentrations of doxorubicin induce a level of oxidative stress in MCF-10A cells that is capable of oxidizing DNA bases and significantly altering cellular proliferation.

Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells.

We investigated the mechanism of action of 2-aminopurine (Apur) in eucaryotic cells. By analogy with studies in procaryotic systems, the base analog is presumed to incorporate into DNA predominantly opposite T where, upon subsequent DNA replication, it can mispair with C, inducing an A:T leads to G:C transition. This model predicts that Apur-induced mutagenesis will be enhanced by factors that favor formation of Apur-C mispairs, e.g., high levels of dCTP or low levels of TTP. We describe the use of a mutant T-lymphosarcoma cell line, AraC-6-1, which has an abnormally high dCTP pool and a low TTP pool, to test this prediction. AraC-6-1 cells were three- to fivefold more mutable by Apur than their parental cell line, NSU-1. This enhanced mutability by Apur could not be explained by altered incorporation of 3H-labeled Apur, by generally impaired ability to repair DNA damage, or by a direct effect of Apur on the endogenous deoxynucleotide pools. The addition of 10 microM thymidine to the growth medium of AraC-6-1 cells lowered their high dCTP pool (two- to threefold), raised the TTP pool (two- to threefold), and abolished their enhanced mutability by Apur. Further manipulation to produce an abnormally high TTP/dCTP ratio suppressed Apur-induced mutagenesis (8- to 10-fold) in both AraC-6-1 and NSU-1 cells. These observations support the hypothesis that Apur induces A:T leads to G:C transitions in mammalian cells by a mispairing mechanism.