FTO regulates the DNA damage response via effects on cell-cycle progression.

The fat mass and obesity-associated protein FTO is an "eraser" of N6-methyladenosine, the most abundant mRNA modification. FTO plays important roles in tumorigenesis. However, its activities have not been fully elucidated and its possible involvement in DNA damage - the early driving event in tumorigenesis - remains poorly characterized. Here, we have investigated the role of FTO in the DNA damage response (DDR) and its underlying mechanisms. We demonstrate that FTO responds to various DNA damage stimuli. FTO is overexpressed in mice following exposure to the promutagens aristolochic acid I and benzo[a]pyrene. Knockout of the FTO gene in TK6 cells, via CRISPR/Cas9, increased genotoxicity induced by DNA damage stimuli (micronucleus and TK mutation assays). Cisplatin- and diepoxybutane-induced micronucleus frequencies and methyl methanesulfonate- and azathioprine-induced TK mutant frequencies were also higher in FTO KO cells. We investigated the potential roles of FTO in DDR. RNA sequencing and enrichment analysis revealed that FTO deletion disrupted the p38 MAPK pathway and inhibited the activation of nucleotide excision repair and cell-cycle-related pathways following cisplatin (DNA intrastrand cross-links) treatment. These effects were confirmed by western blotting and qRT-PCR. FTO deletion impaired cell-cycle arrest at the G2/M phase following cisplatin and diepoxybutane treatment (flow cytometry analysis). Our findings demonstrated that FTO is involved in several aspects of DDR, acting, at least in part, by impairing cell cycle progression.

Analytical technologies to revolutionize the environmental mutagenesis-and genome- research -from the basics to the cutting-edge research-: the Open Symposium of the Japanese Environmental Mutagen and Genome Society (JEMS), 2022.

The Open Symposium of the Japanese Environmental Mutagen and Genome Society (JEMS) entitled "Analytical technologies to revolutionize the environmental mutagenesis and genome research -From the basics to the cutting-edge research-" was held online, on June 11th, 2022. The purpose of this symposium was to provide an opportunity to highlight the cutting-edge research for measurement technologies, and informational and computational (in silico) sciences for the purpose of applying them to deepen scientific knowledge and better understanding the relationship between genes and environmental mutagens. These advanced technologies and sciences are indispensable for the prediction of pharmacokineticses, mutagenicities of chemical substances, and structures of biomolecules including chromosomes. In this symposium, we invited six scientists who are continuing to expand the frontiers in the fields of health data science. Herein, the organizers present a summary of the symposium.

Elevated mutation rates underlie the evolution of the aquatic plant family Podostemaceae.

Molecular evolutionary rates vary among lineages and influence the evolutionary process. Here, we report elevated genome-wide mutation rates in Podostemaceae, a family of aquatic plants with a unique body plan that allows members to live on submerged rocks in fast-flowing rivers. Molecular evolutionary analyses using 1640 orthologous gene groups revealed two historical increases in evolutionary rates: the first at the emergence of the family and the second at the emergence of Podostemoideae, which is the most diversified subfamily. In both branches, synonymous substitution rates were elevated, indicating higher mutation rates. On early branches, mutations were biased in favour of AT content, which is consistent with a role for ultraviolet light-induced mutation and habitat shift. In ancestors of Podostemoideae, DNA-repair genes were enriched in genes under positive selection, which may have responded to the meristem architectural changes.

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair.

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) µ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol µ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol µ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol µ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1-/-/XPA-/-), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1-/-/XPA-/- cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1-/-/XPA-/- cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide.

Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase µ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

Error-prone bypass patch by a low-fidelity variant of DNA polymerase zeta in human cells.

DNA polymerase ζ (Pol ζ) is a specialized Pol that is involved in translesion DNA synthesis (TLS), in particular, in the extension of primer DNA after bypassing DNA lesions. Previously, we established human cells that express a variant form of Pol ζ with an amino acid change of leucine 2618 to methionine (L2618M) in the catalytic subunit REV3L (DNA Repair, 45, 34-43, 2016). This amino acid change made the cells more sensitive to the mutagenicity of benzo[a]pyrene diol epoxide (BPDE). In this study, we embedded BPDE-N2-guanine at a defined position in the supF gene on the shuttle plasmid and introduced it to REV3 L2618M cells or the wild-type (WT) cells to examine how far Pol ζ L2618M extends the primer DNA after bypassing the lesion. The adduct induced primarily G to T and G to C at the adducted site in both cell lines, but generated additional sequence changes such as base substitutions, deletions and additions in the extension patch much more often in REV3 L2618M cells than in the WT cells. Mutations in the extension patch in REV3 L2618M cells occurred most often within 10 bps from the adducted site. Then, the number of mutations gradually decreased and no mutations were observed between 30 and 40 bps from the lesion. We concluded that human Pol ζ L2618M and perhaps WT Pol ζ extend the primer DNA up to approximately 30 bps from the lesion in vivo. The possibility of involvement of Pol ζ L2618M in the insertion step of TLS is discussed.

Tyrosyl-DNA phosphodiesterases are involved in mutagenic events at a ribonucleotide embedded into DNA in human cells.

Ribonucleoside triphosphates are often incorporated into genomic DNA during DNA replication. The accumulation of unrepaired ribonucleotides is associated with genomic instability, which is mediated by DNA topoisomerase 1 (Top1) processing of embedded ribonucleotides. The cleavage initiated by Top1 at the site of a ribonucleotide leads to the formation of a Top1-DNA cleavage complex (Top1cc), occasionally resulting in a DNA double-strand break (DSB). In humans, tyrosyl-DNA phosphodiesterases (TDPs) are essential repair enzymes that resolve the trapped Top1cc followed by downstream repair factors. However, there is limited cellular evidence of the involvement of TDPs in the processing of incorporated ribonucleotides in mammals. We assessed the role of TDPs in mutagenesis induced by a single ribonucleotide embedded into DNA. A supF shuttle vector site-specifically containing a single riboguanosine (rG) was introduced into the human lymphoblastoid TK6 cell line and its TDP1-, TDP2-, and TDP1/TDP2-deficient derivatives. TDP1 and TDP2 insufficiency remarkably decreased the mutant frequency caused by an embedded rG. The ratio of large deletion mutations induced by rG was also substantially lower in TDP1/TDP2-deficient cells than wild-type cells. Furthermore, the disruption of TDPs reduced the length of rG-mediated large deletion mutations. The recovery ratio of the propagated plasmid was also increased in TDP1/TDP2-deficient cells after the transfection of the shuttle vector containing rG. The results suggest that TDPs-mediated ribonucleotide processing cascade leads to unfavorable consequences, whereas in the absence of these repair factors, a more error-free processing pathway might function to suppress the ribonucleotide-induced mutagenesis. Furthermore, base substitution mutations at sites outside the position of rG were detected in the supF gene via a TDPs-independent mechanism. Overall, we provide new insights into the mechanism of mutagenesis induced by an embedded ribonucleotide in mammalian cells, which may lead to the fatal phenotype in the ribonucleotide excision repair deficiency.

Understanding the sequence and structural context effects in oxidative DNA damage repair.

8-Oxo-7,8-dihydroguanine (8-oxoG) is the major base damage in the genomic DNA by exposure to reactive oxygen species. Organisms have evolved various DNA repair mechanisms, such as base excision repair (BER) and nucleotide excision repair (NER), to protect the cellular genome from these mutagenic DNA lesions. The efficiency and capacity of BER and NER mechanisms can be modulated by the local sequence and structural contexts in which 8-oxoG is located. This graphical review summarizes the biochemical and structural studies that have provided insights into the impact of the microenvironment around the site of the lesion on oxidative DNA damage repair.

Processing of a single ribonucleotide embedded into DNA by human nucleotide excision repair and DNA polymerase η.

DNA polymerases often incorporate non-canonical nucleotide, i.e., ribonucleoside triphosphates into the genomic DNA. Aberrant accumulation of ribonucleotides in the genome causes various cellular abnormalities. Here, we show the possible role of human nucleotide excision repair (NER) and DNA polymerase η (Pol η) in processing of a single ribonucleotide embedded into DNA. We found that the reconstituted NER system can excise the oxidized ribonucleotide on the plasmid DNA. Taken together with the evidence that Pol η accurately bypasses a ribonucleotide, i.e., riboguanosine (rG) or its oxidized derivative (8-oxo-rG) in vitro, we further assessed the mutagenic potential of the embedded ribonucleotide in human cells lacking NER or Pol η. A single rG on the supF reporter gene predominantly induced large deletion mutations. An embedded 8-oxo-rG caused base substitution mutations at the 3'-neighboring base rather than large deletions in wild-type cells. The disruption of XPA, an essential factor for NER, or Pol η leads to the increased mutant frequency of 8-oxo-rG. Furthermore, the frequency of 8-oxo-rG-mediated large deletions was increased by the loss of Pol η, but not XPA. Collectively, our results suggest that base oxidation of the embedded ribonucleotide enables processing of the ribonucleotide via alternative DNA repair and damage tolerance pathways.

Comparative study of cytotoxic effects induced by environmental genotoxins using XPC- and CSB-deficient human lymphoblastoid TK6 cells.

BACKGROUND: The human genome is constantly exposed to numerous environmental genotoxicants. To prevent the detrimental consequences induced by the expansion of damaged cells, cellular protective systems such as nucleotide excision repair (NER) exist and serve as a primary pathway for repairing the various helix-distorting DNA adducts induced by genotoxic agents. NER is further divided into two sub-pathways, namely, global genomic NER (GG-NER) and transcription-coupled NER (TC-NER). Both NER sub-pathways are reportedly involved in the damage response elicited by exposure to genotoxins. However, how disruption of these sub-pathways impacts the toxicity of different types of environmental mutagens in human cells is not well understood. RESULTS: To evaluate the role of NER sub-pathways on the cytotoxic effects of mutagens, we disrupted XPC and CSB to selectively inactivate GG-NER and TC-NER, respectively, in human lymphoblastoid TK6 cells, a standard cell line used in genotoxicity studies. Using these cells, we then comparatively assessed their respective sensitivities to representative genotoxic agents, including ultraviolet C (UVC) light, benzo [a] pyrene (B(a)P), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), γ-ray, and 2-acetylaminofluorene (2-AAF). CSB -/- cells exhibited a hyper-sensitivity to UVC, B(a)P, and MeIQx. On the other hand, XPC -/- cells were highly sensitive to UVC, but not to B(a)P and MeIQx, compared with wild-type cells. In contrast with other genotoxins, the sensitivity of XPC -/- cells against PhIP was significantly higher than CSB -/- cells. The toxicity of γ-ray and 2-AAF was not enhanced by disruption of either XPC or CSB in the cells. CONCLUSIONS: Based on our findings, genetically modified TK6 cells appear to be a useful tool for elucidating the detailed roles of the various repair factors that exist to combat genotoxic agents, and should contribute to the improved risk assessment of environmental chemical contaminants.

Current perspectives on mechanisms of ribonucleotide incorporation and processing in mammalian DNA.

Ribonucleotides, which are RNA precursors, are often incorporated into DNA during replication. Although embedded ribonucleotides in the genome are efficiently removed by canonical ribonucleotide excision repair (RER), inactivation of RER causes genomic ribonucleotide accumulation, leading to various abnormalities in cells. Mutation of genes encoding factors involved in RER is associated with the neuroinflammatory autoimmune disorder Aicardi-Goutières syndrome. Over the last decade, the biological impact of ribonucleotides in the genome has attracted much attention. In the present review, we particularly focus on recent studies that have elucidated possible mechanisms of ribonucleotide incorporation and repair and their significance in mammals.

DNA polymerase kappa protects human cells against MMC-induced genotoxicity through error-free translesion DNA synthesis.

BACKGROUND: Interactions between genes and environment are critical factors for causing cancer in humans. The genotoxicity of environmental chemicals can be enhanced via the modulation of susceptible genes in host human cells. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. To better understand the protective roles of Pol κ, we previously engineered two human cell lines either deficient in expression of Pol κ (KO) or expressing catalytically dead Pol κ (CD) in Nalm-6-MSH+ cells and examined cytotoxic sensitivity against various genotoxins. In this study, we set up several genotoxicity assays with cell lines possessing altered Pol κ activities and investigated the protective roles of Pol κ in terms of genotoxicity induced by mitomycin C (MMC), a therapeutic agent that induces bulky DNA adducts and crosslinks in DNA. RESULTS: We introduced a frameshift mutation in one allele of the thymidine kinase (TK) gene of the KO, CD, and wild-type Pol κ cells (WT), thereby establishing cell lines for the TK gene mutation assay, namely TK+/- cells. In addition, we formulated experimental conditions to conduct chromosome aberration (CA) and sister chromatid exchange (SCE) assays with cells. By using the WT TK+/- and KO TK+/- cells, we assayed genotoxicity of MMC. In the TK gene mutation assay, the cytotoxic and mutagenic sensitivities of KO TK+/- cells were higher than those of WT TK+/- cells. MMC induced loss of heterozygosity (LOH), base pair substitutions at CpG sites and tandem mutations at GpG sites in both cell lines. However, the frequencies of LOH and base substitutions at CpG sites were significantly higher in KO TK+/- cells than in WT TK+/- cells. MMC also induced CA and SCE in both cell lines. The KO TK+/- cells displayed higher sensitivity than that displayed by WT TK+/- cells in the SCE assay. CONCLUSIONS: These results suggest that Pol κ is a modulating factor for the genotoxicity of MMC and also that the established cell lines are useful for evaluating the genotoxicity of chemicals from multiple endpoints in different genetic backgrounds of Pol κ.

Mutagenic consequences of cytosine alterations site-specifically embedded in the human genome.

INTRODUCTION: Cytosine residues in CpG dinucleotides often undergo various types of modification, such as methylation, deamination, and halogenation. These types of modifications can be pro-mutagenic and can contribute to the formation of mutational hotspots in cells. To analyze mutations induced by DNA modifications in the human genome, we recently developed a system for tracing DNA adducts in targeted mutagenesis (TATAM). In this system, a modified/damaged base is site-specifically introduced into intron 4 of thymidine kinase genes in human lymphoblastoid cells. To further the understanding of the mutagenesis of cytosine modification, we directly introduced different types of altered cytosine residues into the genome and investigated their genomic consequences using the TATAM system. FINDINGS: In the genome, the pairing of thymine and 5-bromouracil with guanine, resulting from the deamination of 5-methylcytosine and 5-bromocytosine, respectively, was highly pro-mutagenic compared with the pairing of uracil with guanine, resulting from the deamination of cytosine residues. CONCLUSIONS: The deamination of 5-methylcytosine and 5-bromocytosine rather than that of normal cytosine dramatically enhances the mutagenic potential in the human genome.

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine.

Numerous ribonucleotides are incorporated into the genome during DNA replication. Oxidized ribonucleotides can also be erroneously incorporated into DNA. Embedded ribonucleotides destabilize the structure of DNA and retard DNA synthesis by DNA polymerases (pols), leading to genomic instability. Mammalian cells possess translesion DNA synthesis (TLS) pols that bypass DNA damage. The mechanism of TLS and repair of oxidized ribonucleotides remains to be elucidated. To address this, we analyzed the miscoding properties of the ribonucleotides riboguanosine (rG) and 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG) during TLS catalyzed by the human TLS pols κ and η in vitro The primer extension reaction catalyzed by human replicative pol α was strongly blocked by 8-oxo-rG. pol κ inefficiently bypassed rG and 8-oxo-rG compared with dG and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), whereas pol η easily bypassed the ribonucleotides. pol α exclusively inserted dAMP opposite 8-oxo-rG. Interestingly, pol κ preferentially inserted dCMP opposite 8-oxo-rG, whereas the insertion of dAMP was favored opposite 8-oxo-dG. In addition, pol η accurately bypassed 8-oxo-rG. Furthermore, we examined the activity of the base excision repair (BER) enzymes 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 on the substrates, including rG and 8-oxo-rG. Both BER enzymes were completely inactive against 8-oxo-rG in DNA. However, OGG1 suppressed 8-oxo-rG excision by RNase H2, which is involved in the removal of ribonucleotides from DNA. These results suggest that the different sugar backbones between 8-oxo-rG and 8-oxo-dG alter the capacity of TLS and repair of 8-oxoguanine.

Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome.

Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo.

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C.

Translesion DNA synthesis (TLS) is an important pathway that avoids genotoxicity induced by endogenous and exogenous agents. DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in TLS but its protective roles against DNA damage in vivo are still unclear. To better understand these roles, we have established knock-in mice that express catalytically-inactive Polk and crossbred them with gpt delta mice, which possess reporter genes for mutations. The resulting mice (inactivated Polk KI mice) were exposed to mitomycin C (MMC), and the frequency of point mutations, micronucleus formation in peripheral erythrocytes, and γH2AX induction in the bone marrow was determined. The inactivated Polk KI mice exhibited significantly higher frequency of mutations at CpG and GpG sites, micronucleated cells, and γH2AX foci-positive cells than did the Polk wild-type (Polk(+)) mice. Recovery from MMC-induced DNA damage, which was evaluated by γH2AX induction, was retarded in embryonic fibroblasts from the knock-in mice when compared to those from the Polk(+) mice. These results suggest that Polk mediates TLS, which suppresses point mutations and DNA double-strand breaks caused by intra- and interstrand cross-links induced by MMC treatment. The established knock-in mice are extremely useful to elucidate the in vivo roles of the catalytic activity of Polk in suppressing DNA damage that was induced by a variety of genotoxic stresses.

Base excision repair of tandem modifications in a methylated CpG dinucleotide.

Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase ß, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3'-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5'-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase ß activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide.

Role of polymerase ß in complementing aprataxin deficiency during abasic-site base excision repair.

DNA polymerase ß (pol ß) lyase removal of 5'-deoxyribose phosphate (5'-dRP) from base excision repair (BER) intermediates is critical in mammalian BER involving the abasic site. We found that pol ß also removes 5'-adenylated dRP from BER intermediates after abortive ligation. The crystal structure of a human pol ß-DNA complex showed the 5'-AMP-dRP group positioned in the lyase active site. Pol ß expression rescued methyl methanesulfonate sensitivity in aprataxin (hnt3)- and FEN1 (rad27)-deficient yeast.

In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase κ.

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol κ (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N(2)-dG in the supF gene. The frequencies of mutations were in the order of POLK-/->POLK+/->POLK F171A/- both in (-)- and (+)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N(2)-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.