ABSTRACT
Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and proliferating cells cope with accumulating endogenous DNA lesions and how these ultimately affect the physiology of cells and tissues. Here we have addressed these questions by investigating the hematopoietic system of mice deficient for Rev1, a core factor in DNA translesion synthesis (TLS), the postreplicative bypass of damaged nucleotides. Rev1 hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of Xpc, essential for global-genome nucleotide excision repair (ggNER) of helix-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the Rev1Xpc bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of helix-distorting oxidative nucleotide lesions by ggNER and Rev1-dependent TLS, respectively. The error-prone nature of TLS may provide mechanistic understanding of the accumulation of mutations in the hematopoietic system upon aging.
Subject(s)
DNA Damage/genetics , DNA Repair/genetics , Hematopoietic System/physiology , Oxidative Stress , Animals , Apoptosis , Bone Marrow/pathology , Cell Proliferation , Cellular Senescence/genetics , DNA-Directed DNA Polymerase , Genome , Hematopoietic Stem Cells/pathology , Mice , NucleotidyltransferasesABSTRACT
Aristolochic acids (AA) are implicated in the development of chronic renal disease and upper urinary tract carcinoma in humans. Using in vitro approaches, we demonstrated that N-hydroxyaristolactams, metabolites derived from partial nitroreduction of AA, require sulfotransferase (SULT)-catalyzed conjugation with a sulfonyl group to form aristolactam-DNA adducts. Following up on this observation, bioactivation of AA-I and N-hydroxyaristolactam I (AL-I-NOH) was studied in human kidney (HK-2) and skin fibroblast (GM00637) cell lines. Pentachlorophenol, a known SULT inhibitor, significantly reduced cell death and aristolactam-DNA adduct levels in HK-2 cells following exposure to AA-I and AL-I-NOH, suggesting a role for Phase II metabolism in AA activation. A gene knockdown, siRNA approach was employed to establish the involvement of selected SULTs and nitroreductases in AA-I bioactivation. Silencing of SULT1A1 and PAPSS2 led to a significant decrease in aristolactam-DNA levels in both cell lines following exposure to AA-I, indicating the critical role for sulfonation in the activation of AA-I in vivo Since HK-2 cells proved relatively resistant to knockdown with siRNAs, gene silencing of xanthine oxidoreductase, cytochrome P450 oxidoreductase and NADPH:quinone oxidoreductase was conducted in GM00637 cells, showing a significant increase, decrease and no effect on aristolactam-DNA levels, respectively. In GM00637 cells exposed to AL-I-NOH, suppressing the SULT pathway led to a significant decrease in aristolactam-DNA formation, mirroring data obtained for AA-I. We conclude from these studies that SULT1A1 is involved in the bioactivation of AA-I through the sulfonation of AL-I-NOH, contributing significantly to the toxicities of AA observed in vivo.
Subject(s)
Aristolochic Acids/metabolism , Arylsulfotransferase/genetics , Multienzyme Complexes/genetics , Sulfate Adenylyltransferase/genetics , Arylsulfotransferase/antagonists & inhibitors , Arylsulfotransferase/metabolism , Carcinogens/metabolism , Carcinogens/toxicity , DNA/genetics , DNA/metabolism , Fibroblasts/metabolism , Gene Knockdown Techniques , Humans , Kidney/metabolism , Kidney/pathology , Multienzyme Complexes/metabolism , NADPH-Ferrihemoprotein Reductase/metabolism , Pentachlorophenol/pharmacology , RNA, Small Interfering , Sulfate Adenylyltransferase/metabolism , Xanthine Dehydrogenase/metabolismABSTRACT
Aristolochic acid, a potent human carcinogen produced by Aristolochia plants, is associated with urothelial carcinoma of the upper urinary tract (UUC). Following metabolic activation, aristolochic acid reacts with DNA to form aristolactam (AL)-DNA adducts. These lesions concentrate in the renal cortex, where they serve as a sensitive and specific biomarker of exposure, and are found also in the urothelium, where they give rise to a unique mutational signature in the TP53 tumor-suppressor gene. Using AL-DNA adducts and TP53 mutation spectra as biomarkers, we conducted a molecular epidemiologic study of UUC in Taiwan, where the incidence of UUC is the highest reported anywhere in the world and where Aristolochia herbal remedies have been used extensively for many years. Our study involves 151 UUC patients, with 25 patients with renal cell carcinomas serving as a control group. The TP53 mutational signature in patients with UUC, dominated by otherwise rare A:T to T:A transversions, is identical to that observed in UUC associated with Balkan endemic nephropathy, an environmental disease. Prominent TP53 mutational hotspots include the adenine bases of (5')AG (acceptor) splice sites located almost exclusively on the nontranscribed strand. A:T to T:A mutations also were detected at activating positions in the FGFR3 and HRAS oncogenes. AL-DNA adducts were present in the renal cortex of 83% of patients with A:T to T:A mutations in TP53, FGFR3, or HRAS. We conclude that exposure to aristolochic acid contributes significantly to the incidence of UUC in Taiwan, a finding with significant implications for global public health.
Subject(s)
Aristolochic Acids/adverse effects , Carcinoma, Renal Cell/chemically induced , Carcinoma, Transitional Cell/chemically induced , Drugs, Chinese Herbal/adverse effects , Kidney Neoplasms/chemically induced , Ureteral Neoplasms/chemically induced , Adult , Aged , Aged, 80 and over , Carcinoma, Renal Cell/epidemiology , Carcinoma, Renal Cell/genetics , Carcinoma, Transitional Cell/epidemiology , Carcinoma, Transitional Cell/genetics , DNA Adducts/genetics , Female , Humans , Kidney Neoplasms/epidemiology , Kidney Neoplasms/genetics , Male , Middle Aged , Mutagens/adverse effects , Oncogenes/drug effects , Oncogenes/genetics , Taiwan/epidemiology , Tumor Suppressor Protein p53/genetics , Ureteral Neoplasms/epidemiology , Ureteral Neoplasms/genetics , Urothelium/drug effects , Urothelium/pathologyABSTRACT
The emergence of highly infectious pathogens with their potential for triggering global pandemics necessitate the development of effective treatment strategies, including broad-spectrum antiviral therapies to safeguard human health. This study investigates the antiviral activity of emetine, dehydroemetine (DHE), and congeneric compounds against SARS-CoV-2 and HCoV-OC43, and evaluates their impact on the host cell. Concurrently, we assess the potential cardiotoxicity of these ipecac alkaloids. Significantly, our data reveal that emetine and the (-)-R,S isomer of 2,3-dehydroemetine (designated in this paper as DHE4) reduce viral growth at nanomolar concentrations (i.e., IC50 â¼ 50-100 nM), paralleling those required for inhibition of protein synthesis, while calcium channel blocking activity occurs at elevated concentrations (i.e., IC50 â¼ 40-60 µM). Our findings suggest that the antiviral mechanisms primarily involve disruption of host cell protein synthesis and is demonstrably stereoisomer specific. The prospect of a therapeutic window in which emetine or DHE4 inhibit viral propagation without cardiotoxicity renders these alkaloids viable candidates in strategies worthy of clinical investigation.
Subject(s)
Alkaloids , Emetine , Emetine/analogs & derivatives , Humans , Emetine/pharmacology , Ipecac/pharmacology , Cardiotoxicity , Antiviral Agents/toxicityABSTRACT
The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G â T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1(-/-) MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk(-/-) Polh(-/-) Poli(-/-) triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.
Subject(s)
DNA Replication , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Mutagenesis , Nuclear Proteins/metabolism , Nucleotidyltransferases/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/analogs & derivatives , Amino Acid Sequence , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Adducts/genetics , DNA Damage , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/genetics , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/genetics , Humans , Mad2 Proteins , Mice , Mice, Knockout , Molecular Sequence Data , Mutagenesis/drug effects , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/genetics , Protein Binding , Protein Structure, Tertiary , Proteins/genetics , Proteins/metabolism , Sequence AlignmentABSTRACT
Aristolochic acid (AA), a component of all Aristolochia-based herbal medicines, is a potent nephrotoxin and human carcinogen associated with upper urinary tract urothelial carcinoma (UUC). To investigate the clinical and pathological characteristics of AA-induced UUC, this study included 152 UUC patients, 93 of whom had been exposed to AA based on the presence of aristolactam-DNA adducts in the renal cortex. Gene sequencing was used to identify tumors with A:T-to-T:A transversions in TP53, a mutational signature associated with AA. Cases with both aristolactam-DNA adducts and A:T-to-T:A transversions in TP53 were defined as AA-UUC, whereas patients lacking both of these biomarkers were classified as non-AA-UUC. Cases with either biomarker were classified as possible-AA-UUC. Forty (26%), 60 (40%), and 52 (34%) patients were classified as AA-UUC, possible-AA-UUC and non-AA-UUC, respectively. AA-UUC patients were younger (median ages: 64, 68, 68 years, respectively; p=0.189), predominately female (65%, 42%, 35%, respectively; p=0.011), had more end-stage renal disease (28%, 10%, 12%, respectively; p=0.055), and were infrequent smokers (5%, 22%, 33%, respectively; p=0.07) compared to possible-AA-UUC and non-AA-UUC patients. All 14 patients who developed contralateral UUC had aristolactam-DNA adducts; ten of these also had signature mutations. The contralateral UUC-free survival period was shorter in AA-UUC compared to possible- or non-AA-UUC (p=0.019 and 0.002, respectively), whereas no differences among groups were observed for bladder cancer recurrence. In conclusion, AA-UUC patients tend to be younger and female, and have more advanced renal disease. Notably, AA exposure was associated with an increased risk for developing synchronous bilateral and metachronous contralateral UUC.
Subject(s)
Adenine/analogs & derivatives , Aristolochic Acids/adverse effects , Carcinogens , Carcinoma, Transitional Cell/chemically induced , Drugs, Chinese Herbal/adverse effects , Heterocyclic Compounds, 4 or More Rings/metabolism , Mutagens/adverse effects , Mutation , Tumor Suppressor Protein p53/genetics , Urologic Neoplasms/chemically induced , Adenine/metabolism , Adult , Age Factors , Aged , Aged, 80 and over , Carcinoma, Transitional Cell/epidemiology , Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/pathology , DNA Adducts/drug effects , DNA Adducts/metabolism , DNA, Neoplasm/drug effects , DNA, Neoplasm/metabolism , Deoxyadenosines , Drugs, Chinese Herbal/administration & dosage , Female , Humans , Kaplan-Meier Estimate , Kidney Failure, Chronic/etiology , Male , Middle Aged , Neoplasms, Multiple Primary/chemically induced , Neoplasms, Second Primary/chemically induced , Recurrence , Risk Factors , Sequence Analysis, DNA , Sex Factors , Taiwan/epidemiology , Transcriptome , Treatment Outcome , Urologic Neoplasms/epidemiology , Urologic Neoplasms/genetics , Urologic Neoplasms/pathologyABSTRACT
To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochemical studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polymerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N(2)-modified 2'-deoxyguanosine derivatives. Herein, we used shuttle vector technology and demonstrated that deficiency in Polk or Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of GâT and GâA mutations at N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) and N(2)-carboxymethyl-2'-deoxyguanosine (N(2)-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ι preferentially inserts the correct nucleotide, dCMP, opposite N(2)-CEdG lesions. In contrast, no mutation was found after the N(2)-CEdG- and N(2)-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ι are necessary for the error-free bypass of N(2)-CEdG and N(2)-CMdG. However, in the absence of polymerase κ or ι, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately.
Subject(s)
DNA Damage/physiology , DNA Repair/physiology , DNA-Directed DNA Polymerase/metabolism , Deoxyguanosine/metabolism , Animals , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , Deoxyguanosine/genetics , Humans , Mice , DNA Polymerase iotaABSTRACT
Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In approximately 50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA-->T and AL-II-dG-->T transversions.
Subject(s)
Aristolochic Acids/chemical synthesis , DNA Adducts/chemical synthesis , Mutagenesis , Animals , Aristolochic Acids/chemistry , Cells, Cultured , DNA/biosynthesis , DNA Adducts/chemistry , Mice , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistryABSTRACT
This study was designed to establish the TP53 mutational spectrum of aristolochic acid (AA), examined in the context of endemic (Balkan) nephropathy, an environmental disease associated with transitional cell (urothelial) carcinomas of the upper urinary tract (UUC). Tumor tissue was obtained from residents of regions in Bosnia, Croatia and Serbia where endemic nephropathy has been prevalent for over 50 years. Fifty-nine TP53 mutations were detected in 42 of the 97 tumors analyzed. Mutational spectra were dominated by A:T to T:A transversions with the mutated adenines located almost exclusively on the nontranscribed strand. This marked strand bias is attributed to selective processing of aristolactam-dA adducts by transcription-coupled nucleotide excision repair. Hotspots for A:T to T:A mutations include codons 131 and 179 and the 5'-AG acceptor splice site of intron 6. The unique TP53 mutational signature for AA identified in this study can be used to explore the hypothesis that botanical products containing this human carcinogen and nephrotoxin are responsible, in part, for the high prevalence of UUC and chronic renal disease in countries where Aristolochia herbal remedies traditionally have been used for medicinal purposes.
Subject(s)
Aristolochic Acids/toxicity , Carcinogens, Environmental/toxicity , Diet , Genes, p53 , Mutation , Urothelium , Aristolochic Acids/administration & dosage , Carcinoma, Transitional Cell/genetics , HumansABSTRACT
BACKGROUND: Aristolochic acids (AA) and arsenic are chemical carcinogens associated with urothelial carcinogenesis. Here we investigate the combined effects of AA and arsenic toward the risk of developing upper tract urothelial carcinoma (UTUC). METHODS: Hospital-based (n = 89) and population-based (2,921 cases and 11,684 controls) Taiwanese UTUC cohorts were used to investigate the association between exposure to AA and/or arsenic and the risk of developing UTUC. In the hospital cohort, AA exposure was evaluated by measuring aristolactam-DNA adducts in the renal cortex and by identifying A>T TP53 mutations in tumors. In the population cohort, AA exposure was determined from prescription health insurance records. Arsenic levels were graded from 0 to 3 based on concentrations in well water and the presence of arseniasis-related diseases. RESULTS: In the hospital cohort, 43, 26, and 20 patients resided in grade 0, 1+2, and 3 arseniasis-endemic areas, respectively. Aristolactam-DNA adducts were present in >90% of these patients, indicating widespread AA exposure. A>T mutations in TP53 were detected in 28%, 44%, and 22% of patients residing in grade 0, 1+2, and 3 arseniasis-endemic areas, respectively. Population studies revealed that individuals who consumed more AA-containing herbs had a higher risk of developing UTUC in both arseniasis-endemic and nonendemic areas. Logistic regression showed an additive effect of AA and arsenic exposure on the risk of developing UTUC. CONCLUSIONS: Exposure to both AA and arsenic acts additively to increase the UTUC risk in Taiwan. IMPACT: This is the first study to investigate the combined effect of AA and arsenic exposure on UTUC.
Subject(s)
Aristolochic Acids/toxicity , Arsenic/toxicity , Carcinoma, Transitional Cell/chemically induced , Urinary Bladder Neoplasms/chemically induced , Aged , Carcinoma, Transitional Cell/epidemiology , Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/pathology , Case-Control Studies , DNA Adducts , Female , Humans , Incidence , Male , Neoplasm Grading , Taiwan/epidemiology , Urinary Bladder Neoplasms/epidemiology , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
The (6-4) pyrimidine-pyrimidone photoproduct [(6-4)PP] is a major DNA lesion induced by ultraviolet radiation. (6-4)PP induces complex mutations opposite its downstream bases, in addition to opposite 3' or 5' base, as has been observed through a site-specific translesion DNA synthesis (TLS) assay. The mechanism by which these mutations occur is not well understood. To elucidate the mechanisms underlying mutagenesis induced by (6-4)PP, we performed an intracellular TLS assay using a replicative vector with site-specific T(thymidine)-T (6-4)PP. Rev3-/-p53-/- mouse embryonic fibroblast (MEF) cells (defective in Polζ) were almost completely defective in bypassing T-T (6-4)PP, whereas both Rev1-/- and Polh-/-Poli-/-Polk-/- MEF cells (defective in Polη, Polι, and Polκ) presented bypassing activity comparable to that of wild-type cells, indicating that Y-family TLS polymerases are dispensable for bypassing activity, whereas Polζ plays an essential role, probably at the extension step. Among all cells tested, misincorporation occurred most frequently just beyond the lesion (position +1), indicating that the Polζ-dependent extension step is crucial for (6-4)PP-induced mutagenesis. We then examined the effects of sequence context on T-T (6-4)PP bypass using a series of T-T (6-4)PP templates with different sequences at position +1 or -1 to the lesion, and found that the dependency of T-T (6-4)PP bypass on Polζ is not sequence specific. However, the misincorporation frequency at position +1 differed significantly among these templates. The misincorporation of A at position +1 occurred frequently when a purine base was located at position -1. These results indicate that Polζ-dependent extension plays a major role in inducing base substitutions in (6-4)PP-induced mutagenesis, and its fidelity is affected by sequence context surrounding a lesion.
Subject(s)
DNA Damage , Pyrimidine Dimers/metabolism , Animals , DNA/radiation effects , DNA Repair , DNA Replication , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Mice , Mice, Knockout , Mutagenesis , Mutation , Ultraviolet Rays , DNA Polymerase iotaABSTRACT
Reaction of crotonaldehyde or two molecules of acetaldehyde with DNA generates 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)one (2, Scheme 1), which occurs in (6R, 8R) and (6S, 8S) configurations (Fig. 1). These diastereomers were site-specifically incorporated into oligonucleotides, which were then inserted into a double-stranded DNA vector for genotoxicity studies. Modified DNA was introduced into human xeroderma pigmentosum A (XPA) cells to allow replication. Analysis of progeny plasmid revealed that these DNA adducts inhibit DNA synthesis to similar degrees. (6S, 8S)-2 miscodes more frequently than (6R, 8R)-2: 10% versus 5%. For both adducts, major miscoding events were G-->T transversions, but G-->A transitions were also observed at a comparable level for (6R, 8R)-2. G-->C transversions were the second most common events for (6S, 8S)-2. Comparison of these results with those of other 1,N2-propanodeoxyguanosine (PdG) adducts, which were evaluated by the same system, indicates that (i) their synthesis inhibiting potencies are stronger than that of the unsubstituted analog, 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (1, Scheme 1), but weaker than that of 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (3, Scheme 1); (ii) both isomers of 2 are more miscoding than 1; (iii) the miscoding potency of (6S, 8S)-2 is comparable to those of 3 and a model PdG 4 lacking a hydroxyl and a methyl group (Fig. 1). Therefore, considering the fact that 2 are formed endogenously as well as exogenously, they may play a significant role in aging and cancer in humans.
Subject(s)
Acetaldehyde/toxicity , Aldehydes/toxicity , DNA Adducts/toxicity , DNA Damage , Deoxyguanosine/analogs & derivatives , Mutagens , Acetaldehyde/metabolism , Aldehydes/metabolism , Cell Line , DNA Adducts/chemistry , DNA Adducts/metabolism , Deoxyguanosine/chemistry , Deoxyguanosine/metabolism , Humans , Oligonucleotides/chemistry , Oligonucleotides/isolation & purification , Plasmids , Point Mutation , Stereoisomerism , Transfection , Xeroderma Pigmentosum/genetics , Xeroderma Pigmentosum/metabolismABSTRACT
8-oxo-7, 8-dihydrodeoxyguanosine (8-oxo-dG), one of the representative oxidative DNA lesions, frequently mispairs with the incoming dAMP during mammalian DNA replication. Mispaired dA is removed by post-replicative base excision repair (BER) initiated by adenine DNA glycosylase, MYH, creating an apurinic (AP) site. The subsequent mechanism ensuring a dC:8-oxo-dG pair, a substrate for 8-oxoguanine DNA glycosylase (OGG1), remains to be elucidated. At the nucleotide insertion step, none of the mammalian DNA polymerases examined exclusively inserted dC opposite 8-oxo-dG that was located in a gap. AP endonuclease 1, which possesses 3'-->5' exonuclease activity and potentially serves as a proofreader, did not discriminate dA from dC that was located opposite 8-oxo-dG. However, human DNA ligases I and III joined 3'-dA terminus much more efficiently than 3'-dC terminus when paired to 8-oxo-dG. In reconstituted short-patch BER, repair products contained only dA opposite 8-oxo-dG. These results indicate that human DNA ligases discriminate dC from dA and that MYH-initiated short-patch BER is futile and hence this BER must proceed to long-patch repair, even if it is initiated as short-patch repair, through strand displacement synthesis from the ligation-resistant dC terminus to generate the OGG1 substrate, dC:8-oxo-dG pair.
Subject(s)
Adenine/metabolism , Base Pair Mismatch , DNA Repair , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , 8-Hydroxy-2'-Deoxyguanosine , DNA Glycosylases/metabolism , DNA Ligases/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , DNA-Directed DNA Polymerase/metabolism , Deoxyadenine Nucleotides/metabolism , Deoxyadenosines/chemistry , Deoxyadenosines/metabolism , Deoxycytidine/metabolism , Deoxycytidine Monophosphate/metabolism , Deoxyguanosine/chemistry , Exodeoxyribonucleases/metabolism , HumansABSTRACT
Translesion DNA synthesis (TLS) operates when replicative polymerases are blocked by DNA lesions. To investigate the mechanism of mammalian TLS, we employed a plasmid bearing a single 7-(deoxyadenosine-N6-yl)-aristolactam I (dA-AL-I) adduct, which is generated by the human carcinogen, aristolochic acid I, and genetically engineered mouse embryonic fibroblasts. This lesion induces A to T transversions at a high frequency. The simultaneous knockouts of the Polh, Poli and Polk genes did not influence the TLS efficiency or the coding property of dA-AL-I, indicating that an unknown DNA polymerase(s) can efficiently catalyze the insertion of a nucleotide opposite the adduct and subsequent extension. Similarly, knockout of the Rev1 gene did not significantly affect TLS. However, knockout of the Rev3l gene, coding for the catalytic subunit of polζ, drastically suppressed TLS and abolished dA-AL-I to T transversions. The results support the idea that Rev1 is not essential for the cellular TLS functions of polζ in mammalian cells. Furthermore, the frequency of dA-AL-I to T transversion was affected by a sequence context, suggesting that TLS, at least in part, contributes to the formation of mutational hot and cold spots observed in aristolochic acid-induced cancers.
Subject(s)
Aristolochic Acids/pharmacology , Carcinogens/pharmacology , DNA Adducts/biosynthesis , Nucleotidyltransferases/genetics , Animals , Aristolochic Acids/chemistry , Aristolochic Acids/metabolism , Base Sequence , DNA Adducts/chemistry , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Knockout Techniques , Humans , Mice , Nucleotidyltransferases/metabolism , Plasmids/chemistry , Plasmids/metabolism , DNA Polymerase iotaABSTRACT
SLX4 (FANCP) and XPF (FANCQ) proteins interact with each other and play a vital role in the Fanconi anemia (FA) DNA repair pathway. We have identified a SLX4 region and several amino acid residues that are responsible for this interaction. The study has revealed that the global minor allele, SLX4(Y546C), is defective in this interaction and cannot complement Fancp knockout mouse cells in mitomycin C-induced cytotoxicity or chromosomal aberrations. These results highly suggest this allele, as well as SLX4(L530Q), to be pathogenic. The interacting partner XPF is involved in various DNA repair pathways, and certain XPF mutations cause progeria, Cockayne syndrome (CS), and/or FA phenotypes. Because several atypical xeroderma pigmentosum (XP) phenotype-causing XPF missense mutations are located in the SLX4-interacting region, we suspected the disruption of the interaction with SLX4 in these XPF mutants, thereby causing severer phenotypes. The immunoprecipitation assay of cell extracts revealed that those XPF mutations, except XPF(C236R), located in the SLX4-interacting region cause instability of XPF protein, which could be the reason for the FA, progeria and/or CS phenotypes.
Subject(s)
DNA-Binding Proteins/metabolism , Fanconi Anemia/genetics , Mutation, Missense , Recombinases/metabolism , Amino Acid Sequence , Animals , Chromosome Aberrations/chemically induced , Cockayne Syndrome/genetics , DNA Repair , DNA-Binding Proteins/genetics , Fanconi Anemia/metabolism , Genetic Complementation Test , HEK293 Cells , Humans , Mice , Mice, Knockout , Mitomycin/pharmacology , Molecular Sequence Data , Progeria/genetics , Recombinases/genetics , Xeroderma Pigmentosum/geneticsABSTRACT
In humans, exposure to aristolochic acid (AA) is associated with urothelial carcinoma of the upper urinary tract (UTUC). Exome sequencing of UTUCs from 19 individuals with documented exposure to AA revealed a remarkably large number of somatic mutations and an unusual mutational signature attributable to AA. Most of the mutations (72%) in these tumors were A:T-to-T:A transversions, located predominantly on the nontranscribed strand, with a strong preference for deoxyadenosine in a consensus sequence (T/CAG). This trinucleotide motif overlaps the canonical splice acceptor site, possibly accounting for the excess of splice site mutations observed in these tumors. The AA mutational fingerprint was found frequently in oncogenes and tumor suppressor genes in AA-associated UTUC. The AA mutational signature was observed in one patient's tumor from a UTUC cohort without previous indication of AA exposure. Together, these results directly link an established environmental mutagen to cancer through genome-wide sequencing and highlight its power to reveal individual exposure to carcinogens.
Subject(s)
Aristolochic Acids/adverse effects , Exome/genetics , Mutation/drug effects , Mutation/genetics , Sequence Analysis, DNA , Aged , Female , Humans , Male , Middle Aged , Urologic Neoplasms/diagnosis , Urologic Neoplasms/genetics , Urologic Neoplasms/pathology , Urothelium/pathologyABSTRACT
Translesion DNA synthesis (TLS) of damaged DNA templates is catalyzed by specialized DNA polymerases. To probe the cellular TLS mechanism, a host-vector system consisting of mouse fibroblasts and a replicating plasmid bearing a single DNA adduct was developed. This system was used to explore the TLS mechanism of a heptanone-etheno-dC (H-epsilondC) adduct, an endogenous lesion produced by lipid peroxidation. In wild-type cells, H-epsilondC almost exclusively directed incorporation of dT and dA. Whereas knockout of the Y family TLS polymerase genes, Polh, Polk, or Poli, did not qualitatively affect these TLS events, inactivation of the Rev3 gene coding for a subunit of polymerase zeta or of the Rev1 gene abolished TLS associated with dA, but not dT, insertion. The analysis of results of the cellular studies and in vitro TLS studies using purified polymerases has revealed that the insertion of dA and dT was catalyzed by different polymerases in cells. While insertion of dT can be catalyzed by polymerase eta, kappa, and iota, insertion of dA is catalyzed by an unidentified polymerase that cannot catalyze extension from the resulting dA terminus. Therefore, the extension from this terminus requires the activity of polymerase zeta-REV1. These results provide new insight into how cells use different TLS pathways to overcome a synthesis block.
Subject(s)
DNA Adducts/metabolism , DNA Damage , DNA-Directed DNA Polymerase/metabolism , Lipid Peroxidation , Animals , Cell Line, Transformed , DNA Adducts/genetics , DNA-Directed DNA Polymerase/genetics , Gene Knockdown Techniques , MiceABSTRACT
Endemic (Balkan) nephropathy (EN), a devastating renal disease affecting men and women living in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, is characterized by its insidious onset, invariable progression to chronic renal failure and a strong association with transitional cell (urothelial) carcinoma of the upper urinary tract. Significant epidemiologic features of EN include its focal occurrence in certain villages and a familial, but not inherited, pattern of disease. Our experiments test the hypothesis that chronic dietary poisoning by aristolochic acid is responsible for EN and its associated urothelial cancer. Using (32)P-postlabeling/PAGE and authentic standards, we identified dA-aristolactam (AL) and dG-AL DNA adducts in the renal cortex of patients with EN but not in patients with other chronic renal diseases. In addition, urothelial cancer tissue was obtained from residents of endemic villages with upper urinary tract malignancies. The AmpliChip p53 microarray was then used to sequence exons 2-11 of the p53 gene where we identified 19 base substitutions. Mutations at A:T pairs accounted for 89% of all p53 mutations, with 78% of these being A:T --> T:A transversions. Our experimental results, namely, that (i) DNA adducts derived from aristolochic acid (AA) are present in renal tissues of patients with documented EN, (ii) these adducts can be detected in transitional cell cancers, and (iii) A:T --> T:A transversions dominate the p53 mutational spectrum in the upper urinary tract malignancies found in this population lead to the conclusion that dietary exposure to AA is a significant risk factor for EN and its attendant transitional cell cancer.
Subject(s)
Aristolochic Acids/adverse effects , Balkan Nephropathy/chemically induced , Balkan Nephropathy/etiology , Aristolochic Acids/analysis , Aristolochic Acids/chemistry , Carcinoma, Transitional Cell/pathology , DNA Adducts/analysis , DNA Adducts/chemistry , Female , Humans , Kidney/chemistry , Kidney/pathology , Male , Mutation/genetics , Spectrometry, Mass, Electrospray Ionization , Tumor Suppressor Protein p53/genetics , Urologic Neoplasms/pathologyABSTRACT
The repair of acetaldehyde/crotonaldehyde-induced guanine (N2)-guanine (N2) interstrand cross-links (ICLs), 3-(2-deoxyribos-1-yl)-5,6,7,8-(N2-deoxyguanosyl)-6(R or S)-methylpyrimido[1,2-alpha]purine-10(3H)-one, was studied using a shuttle plasmid bearing a site-specific ICL. Since the authentic ICLs can revert to monoadducts, a chemically stable model ICL, 1,3-bis(2'-deoxyguanos-N2-yl)butane derivative, was also employed to probe the ICL repair mechanism. Since the removal of ICL depends on the nucleotide excision repair (NER) mechanism in Escherichia coli, the plasmid bearing the model ICL failed to yield transformants in NER-deficient host cells, proving the stability of this ICL in cells. The authentic ICLs yielded transformants in the NER-deficient hosts; therefore, these transformants are produced by plasmid bearing spontaneously reverted monoadducts. In contrast, in NER-deficient human cells, the model ICL was removed by an NER-independent repair pathway, which is unique to higher eukaryotes. This repair did not associate with a transcriptional event, but with replication. The analysis of repaired molecules revealed that the authentic and model ICLs were repaired mostly (>94%) in an error-free manner in both hosts. The major mutations that were observed were G --> T transversions targeting the cross-linked dG located in the lagging strand template. These results support one of the current models for the mammalian NER-independent ICL repair mechanism, in which a DNA endonuclease(s) unhooks an ICL from the leading strand template at a stalled replication fork site by incising on both sides of the ICL and then translesion synthesis is conducted across the "half-excised" ICL attached to the lagging strand template to restore DNA synthesis.
Subject(s)
Acetaldehyde , Aldehydes , DNA Repair , Escherichia coli/genetics , Guanine/chemistry , Base Sequence , Cross-Linking Reagents , Genetic Vectors , Humans , Molecular Sequence Data , Mutagens , Plasmids , Xeroderma Pigmentosum/geneticsABSTRACT
4-Oxo-2(E)-nonenal, a lipid peroxidation-derived product, reacts with dG, dA, and dC in DNA to form heptanone (H)-etheno (epsilon) adducts. Among the three adducts, H-epsilondC is formed in the greatest abundance in in vitro reactions, and it has been detected in the C57BL/6JAPC(min) mouse model of colorectal cancer. To establish the genotoxic properties of this adduct, a site-specifically modified oligonucleotide was synthesized and incorporated into a shuttle vector. The modified vector was replicated in Escherichia coli and human cells. Analysis of the progeny plasmid has revealed that H-epsilondC strongly blocks DNA synthesis and markedly miscodes in both hosts. The miscoding frequency was 40-50% in bacteria and more than 90% in three human cell lines (xeroderma pigmentosum A and variant cells, and DNA repair wild-type cells). There was a drastic difference in coding events in these two hosts: dG and dC were almost exclusively inserted opposite the lesion in E. coli, while dA and dT were the preferential choices in human cells. These results indicate that this endogenous DNA adduct is very genotoxic to both organisms.