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Effects of siRNA knockdowns of TLS Pols on the replicative bypass of 5S,6R Tg lesion carried on the leading strand template in human cells
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Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stackin...
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... studies have suggested that human TLS Pols can react differently to the 5R,6S vs. 5S,6R isoforms of Tg; for exam- ple, the 5R,6S stereoisomer is less inhibitory to TLS by Polη than the 5S,6R form (7), and Polκ misincorporates a G with a higher frequency opposite the 5R,6S form (11). Hence, we verified whether our conclusion for the combined role of Polκ and Polζ in TLS opposite the 5R,6S form applies also to the 5S,6R form. As shown in Table 2, TLS opposite the 5S,6R form carried on the leading strand template occurs as frequently as opposite the 5R,6S isoform and the various TLS Pols operate very similarly opposite both forms of Tg lesion. Thus, whereas depletion of Polη or Polι has no effect, depletion of either Polκ or Polζ confers an over 50% reduction in TLS frequencies opposite the 5S,6R form as well. ...
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Citations
... Among the four l-nucleoside lesions, l-T exhibited the highest frequency of nucleoside misincorporation in E. coli cells, with l-T → dA (13%), l-T → dG (19%) and l-T → dC (46%) mutations (Supplementary Table S1 and Fig. S12 online). The difference in mutagenic frequencies of l-nucleosides bypass in vivo and in vitro may be attributed to functional interaction between TLS polymerases and other related proteins [33][34][35] . Except that DNA polymerases have relative low error rate on replication, the mutagenic effect may attribute to change of the stereochemistry of the templating nucleotide from D to L, which was expected to alter its interactions with the incoming nucleotide and active site residues and influence nucleotide binding and incorporation 36 . ...
l-nucleosides were the most important antiviral lead compounds because they can inhibit viral DNA polymerase and DNA synthesis of many viruses, whereas they may lead to mutations in DNA replication and cause genomic instability. In this study, we reported the replicative bypass of l-deoxynucleosides in recombinant DNA by restriction enzyme–mediated assays to examine their impact on DNA replication in vitro and in E. coli cells. The results showed that a template l-dC inhibited Taq DNA polymerase reaction, whereas it can be bypassed by Vent (exo⁻) DNA polymerase as well as in cell replication, inserting correct nucleotides opposite l-dC. l-dG can be bypassed by Taq DNA polymerase and in E. coli cells, maintaining insertion of correct incoming nucleotides, and l-dG induced mutagenic replication by Vent (exo⁻) DNA polymerase. In contrast, l-dA can induced mutagenic replication in vitro and in E. coli cells. MD simulations were performed to investigate how DNA polymerase affected replicative bypass and mutations when d-nucleosides replaced with l-nucleosides. This study will provide a basis for the ability to assess the cytotoxic and mutagenic properties of the l-nucleoside drugs.
... Extreme products of pyrimidine oxidation, like urea, confer strong blocks for DNA replication (Imlay, 2003). Some of the oxidized pyrimidines, that maintains an intact ring, such as dihydrothymine, generally do not block the synthesis of DNA and gets freely bypassed by DNA polymerases, whereas, thymine glycol actively blocks the main replicative DNA polymerases in vitro, but is usually bypassed by specialized DNA polymerases (Yoon et al., 2010;Takata et al., 2006). In purines, the chief oxidation product, 8-oxoG is a strong mutagenic abrasion that can pair with cytosine or adenine in vitro conditions (Valavanidis et al., 2009). ...
The present book not only discusses the traditional biomonitoring concepts but also highlights the newly developed biomonitoring techniques.
... However, several chemicals can damage DNA, such as alkylating agents that can generate a broad spectrum of DNA adducts [20][21][22] and oxidizing chemicals that primarily cause base oxidation [23,24]. Consequently, these chemicals can cause deletions [25], base substitutions [26], and physical block to replication forks [27]. The DNA damages may be responsible for the development of cancer [28], chronic degenerative diseases (e.g., atherosclerosis) [29], and neurodegenerative diseases (e. g., Alzheimer's and Parkinson's diseases and metabolic syndrome) [30]. ...
Sulfated polysaccharides (SPs) from seaweeds are potential bioactive natural compounds, but their DNA protective activity is poorly explored. This article aimed to evaluate the genotoxic/antigenotoxic potentials of a sulfated heterofucan from brown seaweed Spatoglossum schröederi (Fucan A – FA) and a sulfated galactan from green seaweed Codium isthomocladum (3G4S) using in vitro Comet assay (alkaline and oxidative versions) with HepG2 cells. The antioxidant activity of these SPs was evaluated by total antioxidant capacity, radical scavenging, metal chelating, and antioxidant enzyme activity assays. Both SPs were not genotoxic. FA and 3G4S displayed strong antigenotoxic activity against oxidizing chemical (H2O2) but not against alkylating chemical (MMS). The DNA damage reduction after a pre-treatment of 72 h with these SPs was 81.42% to FA and 81.38% to 3G4S. In simultaneous exposure to FA or 3G4S with H2O2, HepG2 cells presented 48.04% and 55.41% of DNA damage reduction compared with the control, respectively. The antigenotoxicity of these SPs relates to direct antioxidant activity by blockage of the initiation step of the oxidative chain reaction. Therefore, we conclude that FA and 3G4S could be explored as functional natural compounds with antigenotoxic activity due to their great protection against oxidative DNA damage.
... Among the lesions that stall replication, thymine glycol is known to destabilize the DNA to a significant extent [32][33][34] causing chain termination in vitro and in vivo, leading to cytotoxicity [34][35][36][37][38]. Thymine upon oxidation acquires hydroxyl groups at C5 and C6 position ( Fig. 1C) leading to the formation of a nonplanar thymine glycol lesion in the DNA [6,32]. dATP is predominantly added opposite the lesion; however, subsequent extension beyond the lesion is hindered due to the presence of the methyl group at the C5 position interfering with the base at the 5 0 end [32,39]. Generally non-mutagenic when bypassed by specialized translesion polymerases [39], the thymine glycol lesions have been shown to be potential biomarkers of oxidative DNA damage [40,41]. ...
... dATP is predominantly added opposite the lesion; however, subsequent extension beyond the lesion is hindered due to the presence of the methyl group at the C5 position interfering with the base at the 5 0 end [32,39]. Generally non-mutagenic when bypassed by specialized translesion polymerases [39], the thymine glycol lesions have been shown to be potential biomarkers of oxidative DNA damage [40,41]. These DNA lesions can create strong replication blocks to most replicative DNA polymerases potentially leading to DNA-damage induced apoptosis [42]. ...
... Translesion DNA polymerases such as Polη, Rev1 and Polι employ different strategies to incorporate the correct dCTP nucleotide opposite the 8-oxodG lesion [43][44][45][46]. In eukaryotes, the Tg lesion is bypassed accurately by a combination of the DNA polymerases κ and ζ [39,47]. The addition of nucleotides opposite the lesion by translesion DNA polymerases can be accurate or error-prone, with the latter leading to appearance of deleterious mutations. ...
Apicomplexans such as the malaria parasite Plasmodium falciparum possess a unique organelle known as the apicoplast that has its own circular genome. The apicoplast genome is AT rich and is subjected to oxidative stress from the byproducts of the normal biochemical pathways that operate in the apicoplast. It is expected that oxidative stress will lead to the appearance of DNA lesions such as 2‐hydroxydeoxyadenine, thymine glycol, and 8‐oxodeoxyguanine in the apicoplast genome. The apicoplast genome is replicated by the DNA polymerase activity present in the Pfprex enzyme. We have named the polymerase module of Pfprex as PfpPol and the enzyme belongs to the A family of DNA polymerases. Similar to other members of this family, PfpPol also exhibits high fidelity of DNA synthesis. We show that this enzyme is also capable of carrying out translesion DNA synthesis past common DNA lesions that arise due to oxidative stress. The residues N505 and Y509 from the fingers sub‐domain, which are unique to PfpPol, play an important role in the ability of PfpPol to bypass the three lesions. The observed lesion‐bypass ability of the Pfprex enzyme will minimize the adverse effects of oxidative stress on the apicoplast genome of the malaria parasite. These findings also have implications regarding the evolution of the machinery responsible for replication of organellar genomes.
... Whereas replication through certain DNA lesions can be performed by just one Pol, such as by Pol opposite cyclobutane pyrimidine dimers (CPDs) (1)(2)(3)(4)(5)(6), replication through a vast array of DNA lesions requires the sequential action of two Pols, wherein one Pol inserts a nucleotide (nt) opposite the DNA lesion and another Pol extends synthesis from the inserted nt. Biochemical and structural studies with yeast Pol have provided strong evidence for its role in extending synthesis from nts inserted opposite DNA lesions by other TLS Pols (7)(8)(9)(10), and genetic evidence accrued from TLS studies opposite a number of DNA lesions in human cells aligns with such a Pol role (5,6,11,12). ...
In a previous study we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting a nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ - which normally uses W-C base pairing for DNA synthesis – in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ’s role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively.
... We have shown previously that replication through the Tg adduct is conducted via two alternate TLS pathways dependent upon either Polκ/Polζ or Polθ (Yoon et al, 2010a(Yoon et al, , 2014) (see also Fig 7A). In the Polκ/Polζ pathway, following nt insertion by Polκ opposite the Tg adduct, Polζ would extend synthesis from the nt opposite Tg, and this pathway promotes error-free TLS through the lesion. ...
... In the Polκ/Polζ pathway, following nt insertion by Polκ opposite the Tg adduct, Polζ would extend synthesis from the nt opposite Tg, and this pathway promotes error-free TLS through the lesion. In the alternative pathway, Polθ performs both steps of TLS and it acts in an error-prone manner (Yoon et al, 2010a(Yoon et al, , 2014. As shown in Table 7, TLS opposite Tg occurs at a frequency of~23% in WT MEFs. ...
... Our proposal that after nt insertion by Polκ opposite the Tg lesion, Polζ would extend synthesis (Yoon et al, 2010a) predicted that only the non-catalytic scaffolding role of Polλ would be required for Polζ -dependent TLS opposite Tg. Similarly, the role of Polι in inserting a nt opposite εdA and of Polζ in extending subsequent synthesis (Nair et al, 2006;Yoon et al, 2019a) predicted that only the non-catalytic scaffolding role of Polλ would be required for Polζdependent TLS opposite εdA. ...
By extending synthesis opposite from a diverse array of DNA lesions, DNA polymerase (Pol) ζ performs a crucial role in translesion synthesis (TLS). In yeast and cancer cells, Rev1 functions as an indispensable scaffolding component of Polζ and it imposes highly error-prone TLS upon Polζ. However, for TLS that occurs during replication in normal human cells, Rev1 functions instead as a scaffolding component of Pols η, ι, and κ and Rev1-dependent TLS by these Pols operates in a predominantly error-free manner. The lack of Rev1 requirement for Polζ function in TLS in normal cells suggested that some other protein substitutes for this Rev1 role. Here, we identify a novel role of Polλ as an indispensable scaffolding component of Polζ. TLS studies opposite a number of DNA lesions support the conclusion that as an integral component, Polλ adapts Polζ-dependent TLS to operate in a predominantly error-free manner in human cells, essential for genome integrity and cellular homeostasis.
... Early studies showed that thymine glycols, which comprise up to 30% of the H 2 O 2 -induced base damage, represent strong blocks to E. coli DNA polymerase I and phage T4 DNA polymerase in vitro (Ide et al., 1985;Rouet and Essigmann, 1985;Clark and Beardsley, 1986;Hayes and LeClerc, 1986;Blakely et al., 1990), and it was often inferred that this would extend generally to other DNA polymerases. However, since these initial studies, several other polymerases have been examined and found to efficiently bypass this lesion, including many in humans (Fischhaber et al., 2002;Takata et al., 2006;Belousova et al., 2010;Yoon et al., 2010;Hogg et al., 2011;Makarova et al., 2018). Further, auxiliary proteins associated with the replisome, such as single-strand binding protein and processivity factors, can increase bypass efficiency in some cases (Maga et al., 2007;McCulloch et al., 2009). ...
UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the recF pathway proteins which process and maintain the replication fork DNA to allow the lesion to be repaired before replication resumes. Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions.
... In higher eukaryotes, a number of TLS polymerases have been implicated in Tg bypass (Fischhaber et al, 2002;Kusumoto et al, 2002;Takata et al, 2006;Belousova et al, 2010;Yoon et al, 2010Yoon et al, , 2014, many of which are absent in S. cerevisiae. Yeast Pol d may therefore possess an increased ability to tolerate Tg due to the lack of these additional polymerases. ...
The high-fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low-fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading-strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase-polymerase uncoupling a switch from Pol ε, the canonical leading-strand replicase, to the lagging-strand replicase Pol δ, facilitates rapid, efficient and error-free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8-oxoguanine. We propose that replicase switching may promote continued leading-strand synthesis whenever the replisome encounters leading-strand damage that is bypassed more efficiently by Pol δ than by Pol ε.
... As the carrier of genetic information, DNA's chemical integrity has to be maintained for normal cellular functions. Cells are continuously exposed to exogenous and endogenous genotoxic agents, which give rise to numerous DNA lesions on a daily basis (1 Although some TLS Pols can efficiently and accurately bypass specific DNA lesions (5,6), this damage tolerance mechanism is often error-prone (7,8), which may confer increased risks of mutagenesis and carcinogenesis (9). For instance, human Pol has been shown to accurately and efficiently bypass cyclobutane thymine dimer (10); ...
Exogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here, we examined the functions of both polymerases in bypassing the major-groove O⁶ -alkyl-2′-deoxyguanosine ( O⁶ -alkyl-dG) and the minor-groove N² -alkyl-dG lesions in human cells, where the alkyl groups are ethyl (Et), n -butyl (nBu), and, for O⁶ -alkyl-dG, pyridyloxobutyl (POB). We found that Pol ν and Pol θ promote TLS across the major-groove O⁶ -alkyl-dG lesions. The O⁶ -alkyl-dG lesions mainly induced G->A mutations, which were modulated by the two TLS polymerases and the structures of the alkyl groups. Simultaneous ablation of Pol ν and Pol θ resulted in diminished mutation frequencies for all three O⁶ -alkyl-dG lesions, depletion of Pol ν alone reduced mutations only for O⁶ -nBu-dG, and sole loss of Pol θ attenuated the mutation rates for both O⁶ - n Bu-dG and O⁶ -POB-dG. Replication across the two N² -alkyl-dG lesions was error-free, and Pol ν and Pol θ were dispensable for their replicative bypass. Together, our results provide critical knowledge about the involvement of Pol ν and Pol θ in bypassing alkylated guanine lesions in human cells.
... 42 In contrast, Tg is known to be only weakly mutagenic in mammalian cells. 43 When L is part of a tandem lesion with a Tg located 3' to it (5'-LTg), the nucleotide incorporation pattern opposite it is not significantly affected in E. coli. 28 However, in addition to single-base deletions, varying levels of three-base deletions were observed in TLS polymerase-deficient cells. ...
... In pol ζ KO cells, nearly equal frequency of targeted and semi-targeted mutations was observed. In a previous study of Tg mutagenicity in human cells, it was postulated that error-free bypass of Tg is carried out by the combined action of pol κ and pol ζ. 43 Therefore, we also evaluated Tg mutagenesis in pol ζ KO cells after knocking down pol κ with synthetic siRNA. Indeed, MF of Tg increased by ~70% (to 5.5%), but a significant fraction of mutants involved semi-targeted mutations, as noted in pol ζdeficient cells. ...
... Despite the increased MF in pol κand pol κ/pol ζ-deficient cells, some of our results are different from the previous study, 43 which could be due to the differences in cell type (HEK 293T versus human fibroblast), single-stranded versus duplex vector, and the DNA sequence context used in the two investigations. ...
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) and thymine glycol (Tg), and the 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. 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 MF of Tg (to ~5.5%) was observed in cells with deficiency of 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, dA was preferentially incorporated opposite an isolated L, as well as when it was part of a tandem lesion, in all cell types. 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%, respectively, in pol κ-, pol ζ- and pol ι-deficient cells. 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.
