5-Formylcytosine mediated DNA-peptide cross-link induces predominantly semi-targeted mutations in both Escherichia coli and human cells.

Histone proteins can become trapped on DNA in the presence of 5-formylcytosine (5fC) to form toxic DNA-protein conjugates. Their repair may involve proteolytic digestion resulting in DNA-peptide cross-links (DpCs). Here, we have investigated replication of a model DpC comprised of an 11-mer peptide (NH2-GGGKGLGK∗GGA) containing an oxy-lysine residue (K∗) conjugated to 5fC in DNA. Both CXG and CXT (where X = 5fC-DpC) sequence contexts were examined. Replication of both constructs gave low viability (<10%) in Escherichia coli, whereas TLS efficiency was high (72%) in HEK 293T cells. In E. coli, the DpC was bypassed largely error-free, inducing only 2 to 3% mutations, which increased to 4 to 5% with SOS. For both sequences, semi-targeted mutations were dominant, and for CXG, the predominant mutations were G→T and G→C at the 3'-base to the 5fC-DpC. In HEK 293T cells, 7 to 9% mutations occurred, and the dominant mutations were the semi-targeted G → T for CXG and T → G for CXT. These mutations were reduced drastically in cells deficient in hPol η, hPol ι or hPol ζ, suggesting a role of these TLS polymerases in mutagenic TLS. Steady-state kinetics studies using hPol η confirmed that this polymerase induces G → T and T → G transversions at the base immediately 3' to the DpC. This study reveals a unique replication pattern of 5fC-conjugated DpCs, which are bypassed largely error-free in both E. coli and human cells and induce mostly semi-targeted mutations at the 3' position to the lesion.

8-OxodGuo and Fapy•dG Mutagenicity in Escherichia coli Increases Significantly when They Are Part of a Tandem Lesion with 5-Formyl-2'-deoxyuridine.

Tandem lesions, which are defined by two or more contiguously damaged nucleotides, are a hallmark of ionizing radiation. Recently, tandem lesions containing 5-formyl-2'-deoxyuridine (5-fdU) flanked by a 5'-8-OxodGuo or Fapy•dG were discovered, and they are more mutagenic in human cells than the isolated lesions. In the current study, we examined replication of these tandem lesions in Escherichia coli. Bypass efficiency of both tandem lesions was reduced by 30-40% compared to the isolated lesions. Mutation frequencies (MFs) of isolated 8-OxodGuo and Fapy•dG were low, and no mutants were isolated from replication of a 5-fdU construct. The types of mutations from 8-OxodGuo were targeted G → T transversion, whereas Fapy•dG predominantly gave G → T and G deletion. 5'-8-OxodGuo-5-fdU also gave exclusively G → T mutation, which was 3-fold and 11-fold greater, without and with SOS induction, respectively, compared to that of an isolated 8-OxodGuo. In mutY/mutM cells, the MF of 8-OxodGuo and 5'-8-OxodGuo-5-fdU increased 13-fold and 7-fold, respectively. The MF of 5'-8-OxodGuo-5-fdU increased 2-fold and 3-fold in Pol II- and Pol IV-deficient cells, respectively, suggesting that these polymerases carry out largely error-free bypass. The MF of 5'- Fapy•dG-5-fdU was similar without (13 ± 1%) and with (16 ± 2%) SOS induction. Unlike the complex mutation spectrum reported earlier in human cells for 5'- Fapy•dG-5-fdU, with G → T as the major type of errors, in E. coli, the mutations were predominantly from deletion of 5-fdU. We postulate that removal of adenine-incorporated opposite 8-OxodGuo by Fpg and MutY repair proteins is partially impaired in the tandem 5'-8-OxodGuo-5-fdU, resulting in an increase in the G → T mutations, whereas a slippage mechanism may be operating in the 5'- Fapy•dG-5-fdU mutagenesis. This study showed that not only are these tandem lesions more mutagenic than the isolated lesions but they may also exhibit different types of mutations in different organisms.

8-Oxo-2'-deoxyguanosine Replication in Mutational Hot Spot Sequences of the p53 Gene in Human Cells Is Less Mutagenic than That of the Corresponding Formamidopyrimidine.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-OxodGuo) is a ubiquitous DNA damage formed by oxidation of 2'-deoxyguanosine. In this study, plasmid DNA containing 8-OxodGuo located in three mutational hot spots of human cancers, codons 248, 249, and 273 of the Tp53 tumor suppressor gene, was replicated in HEK 293T cells. 8-OxodGuo was only a weak block of replication, and the bypass was largely error-free. The mutations (1-5%) were primarily G → T transversions, and the mutation frequency was generally lower than that of the chemically related Fapy·dG. A unique 8-OxodGuo mutation spectrum was observed at each site, as reflected by replication in translesion synthesis (TLS) polymerase- or hPol λ-deficient cells. In codon 248 (CG*G) and 249 (AG*G), where G* denotes 8-OxodGuo, hPol η and hPol ζ carried out largely error-free bypass of the lesion, whereas hPol κ and hPol ι were involved mostly in error-prone TLS, resulting in G → T mutations. 8-OxodGuo bypass in codon 273 (CG*T) was unlike the other two sites, as hPol κ participated in the mostly error-free bypass of the lesion. Yet, in all three sites, including codon 273, simultaneous deficiency of hpol κ and hPol ι resulted in reduction of G → T transversions. This indicates a convincing role of these two TLS polymerases in error-prone bypass of 8-OxodGuo. Although the dominant mutation was G → T in each site, in codon 249, and to a lesser extent in codon 248, significant semi-targeted single-base deletions also occurred, which suggests that 8-OxodGuo can initiate slippage of a base near the lesion site. This study underscores the importance of sequence context in 8-OxodGuo mutagenesis in human cells. It also provides a more comprehensive comparison between 8-OxodGuo and the sister lesion, Fapy·dG. The greater mutagenicity of the latter in the same sequence contexts indicates that Fapy·dG is a biologically significant lesion and biomarker on par with 8-OxodGuo.

Structural Dynamics of a Common Mutagenic Oxidative DNA Lesion in Duplex DNA and during DNA Replication.

N6-(2-Deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido pyrimidine (Fapy•dG) is a prevalent form of genomic DNA damage. Fapy•dG is formed in greater amounts under anoxic conditions than the well-studied, chemically related 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo). Fapy•dG is more mutagenic in mammalian cells than 8-oxodGuo. A distinctive property of Fapy•dG is facile epimerization, but prior works with Fapy•dG analogues have precluded determining its effect on chemistry. We present crystallographic characterization of natural Fapy•dG in duplex DNA and as the template base for DNA polymerase ß (Pol ß). Fapy•dG adopts the ß-anomer when base paired with cytosine but exists as a mixture of α- and ß-anomers when promutagenically base paired with adenine. Rotation about the bond between the glycosidic nitrogen atom and the pyrimidine ring is also affected by the opposing nucleotide. Sodium cyanoborohydride soaking experiments trap the ring-opened Fapy•dG, demonstrating that ring opening and epimerization occur in the crystalline state. Ring opening and epimerization are facilitated by propitious water molecules that are observed in the structures. Determination of Fapy•dG mutagenicity in wild type and Pol ß knockdown HEK 293T cells indicates that Pol ß contributes to G → T transversions but also suppresses G → A transitions. Complementary kinetic studies have determined that Fapy•dG promotes mutagenesis by decreasing the catalytic efficiency of dCMP insertion opposite Fapy•dG, thus reducing polymerase fidelity. Kinetic studies have determined that dCMP incorporation opposite the ß-anomer is ∼90 times faster than the α-anomer. This research identifies the importance of anomer dynamics, a feature unique to formamidopyrimidines, when considering the incorporation of nucleotides opposite Fapy•dG and potentially the repair of this structurally unusual lesion.

Establishing Linkages Among DNA Damage, Mutagenesis, and Genetic Diseases.

DNA damage by chemicals, radiation, or oxidative stress leads to a mutational spectrum, which is complex because it is determined in part by lesion structure, the DNA sequence context of the lesion, lesion repair kinetics, and the type of cells in which the lesion is replicated. Accumulation of mutations may give rise to genetic diseases such as cancer and therefore understanding the process underlying mutagenesis is of immense importance to preserve human health. Chemical or physical agents that cause cancer often leave their mutational fingerprints, which can be used to back-calculate the molecular events that led to disease. To make a clear link between DNA lesion structure and the mutations a given lesion induces, the field of single-lesion mutagenesis was developed. In the last three decades this area of research has seen much growth in several directions, which we attempt to describe in this Perspective.

Mutagenic Effects of a 2-Deoxyribonolactone-Thymine Glycol Tandem DNA Lesion in Human Cells.

Tandem DNA lesions containing two contiguously damaged nucleotides are commonly formed by ionizing radiation. Their effects on replication in mammalian cells are largely unknown. Replication of isolated 2-deoxyribonolactone (L), thymine glycol (Tg), and tandem lesion 5'-LTg was examined in human cells. Although nearly 100% of Tg was bypassed in HEK 293T cells, L was a significant replication block. 5'-LTg was an even stronger replication block with 5% TLS efficiency. The mutation frequency (MF) of Tg was 3.4%, which increased to 3.9% and 4.8% in pol ι- and pol κ-deficient cells, respectively. An even greater increase in the MF of Tg (to ∼5.5%) was observed in cells deficient in both pol κ and pol ζ, suggesting that they work together to bypass Tg in an error-free manner. Isolated L bypass generated 12-18% one-base deletions, which increased as much as 60% in TLS polymerase-deficient cells. The fraction of deletion products also increased in TLS polymerase-deficient cells upon 5'-LTg bypass. In full-length products and in all cell types, dA was preferentially incorporated opposite an isolated L as well as when it was part of a tandem lesion. However, misincorporation opposite Tg increased significantly when it was part of a tandem lesion. In wild type cells, targeted mutations increased about 3-fold to 9.7% and to 17.4, 15.9, and 28.8% in pol κ-, pol ζ-, and pol ι-deficient cells, respectively. Overall, Tg is significantly more miscoding as part of a tandem lesion, and error-free Tg replication in HEK 293T cells requires participation of the TLS polymerases.

Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells.

DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a site-specific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C→T transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPol ι or hPol ζ reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol η and hPol κ with knockdown of hPol ζ, suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol δ and hPol ϵ can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases.

6-Nitrochrysene-Derived C8-2'-Deoxyadenosine Adduct: Synthesis of Site-Specific Oligodeoxynucleotides and Mutagenicity in Escherichia coli.

6-Nitrochrysene (6-NC), the most potent carcinogen evaluated by the newborn mouse assay, is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields three major DNA adducts: at the C8 and N2 positions of 2'-deoxyguanosine (dG), N-(dG-8-yl)-6-AC and 5-(dG-N2-yl)-6-AC, and at the C8 position of 2'-deoxyadenosine (dA), N-(dA-8-yl)-6-AC. A nucleotide excision repair assay demonstrated that N-(dA-8-yl)-6-AC is repaired much more slowly than many other bulky DNA adducts, including the other DNA adducts formed by 6-NC. But neither the total synthesis nor evaluation of other biological activities of this dA adduct has ever been reported. Herein, we report a convenient synthesis of the 6-NC-derived dA adduct by employing the Buchwald-Hartwig coupling strategy, which provided a high yield of the protected N-(dA-8-yl)-6-AC. The deprotected nucleoside showed syn conformational preference by NMR spectroscopy. Following DMT protection of the 5'-hydroxyl, N-(dA-8-yl)-6-AC was converted to its 3'-phosphoramidite, which was used to prepare oligonucleotides containing a single N-(dA-8-yl)-6-AC adduct. Circular dichroism spectra of the adducted duplex showed only a slight departure from the B-DNA helix profile of the control duplex. The 15-mer N-(dA-8-yl)-6-AC oligonucleotide was used to construct a single-stranded plasmid vector containing a single adduct, which was replicated in Escherichia coli. Viability of the adducted construct was ∼60% of the control, indicating slower translesion synthesis of the adduct, which increased to nearly 90% upon induction of the SOS functions. Without SOS, the mutation frequency (MF) of the adduct was 5.2%, including 2.9% targeted and 2.3% semi-targeted mutations. With SOS, the targeted MF increased 3-fold to 9.0%, whereas semi-targeted mutation increased only marginally to 3.2%. The major type of targeted mutation was A*→G in both uninduced and SOS-induced cells.

Site-Specific Incorporation of N-(2'-Deoxyguanosine-8-yl)-6-aminochrysene Adduct in DNA and Its Replication in Human Cells.

The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen and a mammary carcinogen in rats. 6-NC is the most potent carcinogen ever tested in the newborn mouse assay. In mammalian cells, it is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields two major adducts with 2'-deoxyguanosine (dG), one at the C8-position, N-(dG-8-yl)-6-AC, and the other at the exocyclic N2-position, 5-(dG-N2-yl)-6-AC. Here, we report the total synthesis of a site-specific oligonucleotide containing the 6-NC-derived C8 dG adduct, N-(dG-8-yl)-6-AC. Pd-catalyzed Buchwald-Hartwig cross coupling of 6-aminochrysene with protected C8-bromo-dG derivative served as the key reaction to furnish protected N-(dG-8-yl)-6-AC in 56% yield. The monomer for solid-phase DNA synthesis was prepared by its deprotection followed by conversion to the corresponding 5'-O-dimethoxytrityl 3'-phosphoramidite, which was used to synthesize a site-specifically adducted oligonucleotide. After purification and characterization, the adduct-containing oligonucleotide was incorporated into a plasmid and replicated in human embryonic kidney (HEK) 293T cells, which showed that N-(dG-8-yl)-6-AC stalls DNA replication as evidenced by 77% translesion synthesis (TLS) efficiency relative to the control and that the adduct is mutagenic (mutation frequency (MF) 17.8%) inducing largely G→T transversions. We also investigated the roles of several translesion synthesis DNA polymerases in the bypass of N-(dG-8-yl)-6-AC using siRNA knockdown approach. TLS efficiency was reduced in hPol η-, hPol κ-, hPol ζ-, and hREV1-deficient HEK 293T cells to 66%, 45%, 37%, and 32%, respectively. Notably, TLS efficiency was reduced to 18% in cells with concurrent knockdown of hPol κ, hPol ζ, and REV1, suggesting that these three polymerases play critical roles in bypassing N-(dG-8-yl)-6-AC. MF increased to 23.1% and 32.2% in hPol κ- and hREV1-deficient cells, whereas it decreased to 11.8% in hPol ζ-deficient cells. This suggests that hPol κ and hREV1 are involved in error-free TLS of this lesion, whereas hPol ζ performs error-prone bypass.

5-Formylcytosine mediated DNA-protein cross-links block DNA replication and induce mutations in human cells.

5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA-protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130-14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases η and κ. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and -1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases η and κ revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.

DNA Damage, Mutagenesis and Cancer.

A large number of chemicals and several physical agents, such as UV light and γ-radiation, have been associated with the etiology of human cancer. Generation of DNA damage (also known as DNA adducts or lesions) induced by these agents is an important first step in the process of carcinogenesis. Evolutionary processes gave rise to DNA repair tools that are efficient in repairing damaged DNA; yet replication of damaged DNA may take place prior to repair, particularly when they are induced at a high frequency. Damaged DNA replication may lead to gene mutations, which in turn may give rise to altered proteins. Mutations in an oncogene, a tumor-suppressor gene, or a gene that controls the cell cycle can generate a clonal cell population with a distinct advantage in proliferation. Many such events, broadly divided into the stages of initiation, promotion, and progression, which may occur over a long period of time and transpire in the context of chronic exposure to carcinogens, can lead to the induction of human cancer. This is exemplified in the long-term use of tobacco being responsible for an increased risk of lung cancer. This mini-review attempts to summarize this wide area that centers on DNA damage as it relates to the development of human cancer.

Translesion Synthesis of 2'-Deoxyguanosine Lesions by Eukaryotic DNA Polymerases.

With the discovery of translesion synthesis DNA polymerases, great strides have been made in the last two decades in understanding the mode of replication of various DNA lesions in prokaryotes and eukaryotes. A database search indicated that approximately 2000 articles on this topic have been published in this period. This includes research involving genetic and structural studies as well as in vitro experiments using purified DNA polymerases and accessory proteins. It is a daunting task to comprehend this exciting and rapidly emerging area of research. Even so, as the majority of DNA damage occurs at 2'-deoxyguanosine residues, this perspective attempts to summarize a subset of this field, focusing on the most relevant eukaryotic DNA polymerases responsible for their bypass.

Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases.

DNA-protein cross-links are formed upon exposure of cellular DNA to various agents, including antitumor drugs, UV light, transition metals, and reactive oxygen species. They are thought to contribute to cancer, aging, and neurodegenerative diseases. It has been proposed that DNA-protein cross-links formed in cells are subject to proteolytic degradation to the corresponding DNA-peptide cross-links (DpCs). To investigate the effects of DpCs on DNA replication, we have constructed plasmid DNA containing a 10-mer Myc peptide covalently linked to C7 of 7-deaza-dG, a hydrolytically stable mimic of N7-dG lesions. Following transfection in human embryonic kidney cells (HEK 293T), progeny plasmids were recovered and sequenced. Translesion synthesis (TLS) past DpC was 76% compared to that of the unmodified control. The DpC induced 20% targeted G → A and G → T plus 15% semitargeted mutations, notably at a guanine (G5) five bases 3' to the lesion site. Proteolytic digestion of the DpC reduced the mutation frequency considerably, indicating that the covalently attached 10-mer peptide was responsible for the observed mutations. TLS efficiency and targeted mutations were reduced upon siRNA knockdown of pol η, pol κ, or pol ζ, indicating that they participate in error-prone bypass of the DpC lesion. However, the semitargeted mutation at G5 was only reduced upon knockdown of pol ζ, suggesting its critical role in this type of mutations. Our results indicate that DpCs formed at the N7 position of guanine can induce both targeted and semitargeted mutations in human cells and that the TLS polymerases play a critical role in their error-prone bypass.

Controlling the Graphene-Bio Interface: Dispersions in Animal Sera for Enhanced Stability and Reduced Toxicity.

Liquid phase exfoliation of graphite in six different animal sera and evaluation of its toxicity are reported here. Previously, we reported the exfoliation of graphene using proteins, and here we extend this approach to complex animal fluids. A kitchen blender with a high-turbulence flow gave high quality and maximum exfoliation efficiency in all sera tested, when compared to the values found with shear and ultrasonication methods. Raman spectra and electron microscopy confirmed the formation of three- or four-layer, submicrometer size graphene, independent of the serum used. Graphene prepared in serum was directly transferred to cell culture media without post-treatments. Contrary to many reports, a nanotoxicity study of this graphene fully dispersed to human embryonic kidney cells, human lung cancer cells, and nematodes (Caenorhabditis elegans) showed no acute toxicity for up to 7 days at various doses (50-500 µg/mL), but prolonged exposure at higher doses (300-500 µg/mL, 10-15 days) showed cytotoxicity to cells (∼95% death) and reproductive toxicity to C. elegans (5-10% reduction in brood size). The origin of toxicity was found to be due to the highly fragmented smaller graphene sheets (<200 nm), while the larger sheets were nontoxic (50-300 µg/mL dose). In contrast, graphene produced with sodium cholate as the mediator has been found to be cytotoxic to these cells at these dosages. We demonstrated the toxicity of liquid phase exfoliated graphene is attributed to highly fragmented fractions or nonbiocompatible exfoliating agents. Thus, low-toxicity graphene/serum suspensions are produced by a facile method in biological media, and this approach may accelerate the much-anticipated development of graphene for biological applications.

DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8-2'-deoxyguanosine adduct of the dietary mutagen IQ in human cells.

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8-2'-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G1-, G2- or G3-positions of the NarI site (5'-G1G2CG3CC-3') were replicated in HEK293T cells. Fifty percent of the progeny from the G3 construct were mutants, largely G→T, compared to 18% and 24% from the G1 and G2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38-67% upon siRNA knockdown of pol κ, whereas it was increased by 10-24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G1 and G3 constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G2 construct. In vitro TLS using yeast pol ζ showed that it can extend G3*:A pair more efficiently than G3*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.

Mechanism of Error-Free Bypass of the Environmental Carcinogen N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone Adduct by Human DNA Polymerase†η.

The environmental pollutant 3-nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8-dG-ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error-free manner by the human Y-family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol⋅DNA⋅dCTP complex between a C8-dG-ABA-containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error-free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra-nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson-Crick pair with the incoming dCTP.

Translesion Synthesis of the N(2)-2'-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error-Free Replication by DNA Polymerase κ and Mutagenic Bypass by DNA Polymerases η, ζ, and Rev1.

Translesion synthesis (TLS) of the N(2)-2'-deoxyguanosine (dG-N(2)-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5'-CG1G2CG3CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N(2)-IQ located at G1, G2, and G3, respectively, which suggests that dG-N(2)-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8-35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N(2)-IQ bypass. Mutation frequencies (MFs) of dG-N(2)-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ( ( 2015 ) Nucleic Acids Res. 43 , 8340 - 8351 ). The major type of mutation induced by dG-N(2)-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N(2)-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.

Comparative Error-Free and Error-Prone Translesion Synthesis of N(2)-2'-Deoxyguanosine Adducts Formed by Mitomycin C and Its Metabolite, 2,7-Diaminomitosene, in Human Cells.

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates DNA upon reductive activation. 2,7-Diaminomitosene (2,7-DAM) is a major metabolite of MC in tumor cells, which also alkylates DNA. MC forms seven DNA adducts, including monoadducts and inter- and intrastrand cross-links, whereas 2,7-DAM forms two monoadducts. Herein, the biological effects of the dG-N(2) adducts formed by MC and 2,7-DAM have been compared by constructing single-stranded plasmids containing these adducts and replicating them in human embryonic kidney 293T cells. Translesion synthesis (TLS) efficiencies of dG-N(2)-MC and dG-N(2)-2,7-DAM were 38 ± 3 and 27 ± 3%, respectively, compared to that of a control plasmid. This indicates that both adducts block DNA synthesis and that dG-N(2)-2,7-DAM is a stronger replication block than dG-N(2)-MC. TLS of each adducted construct was reduced upon siRNA knockdown of pol η, pol κ, or pol ζ. For both adducts, the most significant reduction occurred with knockdown of pol κ, which suggests that pol κ plays a major role in TLS of these dG-N(2) adducts. Analysis of the progeny showed that both adducts were mutagenic, and the mutation frequencies (MF) of dG-N(2)-MC and dG-N(2)-2,7-DAM were 18 ± 3 and 10 ± 1%, respectively. For both adducts, the major type of mutation was G → T transversions. Knockdown of pol η and pol ζ reduced the MF of dG-N(2)-MC and dG-N(2)-2,7-DAM, whereas knockdown of pol κ increased the MF of these adducts. This suggests that pol κ predominantly carries out error-free TLS, whereas pol η and pol ζ are involved in error-prone TLS. The largest reduction in MF by 78 and 80%, respectively, for dG-N(2)-MC and dG-N(2)-2,7-DAM constructs occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down. This result strongly suggests that, unlike pol κ, these three TLS polymerases cooperatively perform the error-prone TLS of these adducts.

Unlike catalyzing error-free bypass of 8-oxodGuo, DNA polymerase λ is responsible for a significant part of Fapy·dG-induced G → T mutations in human cells.

8-OxodGuo and Fapy·dG induced 10-22% mutations, predominantly G → T transversions, in human embryonic kidney 293T cells in four TG*N sequence contexts, where N = C, G, A, or T. siRNA knockdown of pol λ resulted in 34 and 55% increases in the level of mutations in the progeny from the 8-oxodGuo construct in the TG*T and TG*G sequences, respectively, suggesting that pol λ is involved in error-free bypass of 8-oxodGuo. For Fapy·dG, in contrast, the level of G → T mutations was reduced by 27 and 46% in the TG*T and TG*G sequences, respectively, suggesting that pol λ is responsible for a significant fraction of Fapy·dG-induced G → T mutations.

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase.

1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N(2)-yl)-1-aminopyrene (dG(1,8)), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG(1,8) bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG(1,8), we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG(1,8) lesion in the absence or presence of dCTP. The Dpo4·DNA-dG(1,8) binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG(1,8)·dCTP ternary structure, the aminopyrene moiety of the dG(1,8) lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson-Crick base pair with dG, two nucleotides upstream from the dG(1,8) site, creating a complex for "-2" frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.