The roles of polymerases ν and θ in replicative bypass of O 6- and N 2-alkyl-2'-deoxyguanosine lesions in human cells.
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ABSTRACT:
SUBMITTER: Du H
PROVIDER: S-EPMC7135994 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 ?, 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 G?T and G?A 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 ? 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 ? and ? are necessary for the error-free bypass of N(2)-CEdG and N(2)-CMdG. However, in the absence of polymerase ? or ?, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately.
| S-EPMC3093824 | biostudies-other
Project description:Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O 6-alkyl-2'-deoxyguanosine (O 6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O 6-alkyl-dG lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, or sBu, and examined the impact of these lesions on DNA replication in Escherichia coli cells. We found that replication past all the O 6-alkyl-dG lesions was highly efficient and that SOS-induced DNA polymerases play redundant roles in bypassing these lesions. Moreover, these lesions directed exclusively the G ? A mutation, the frequency of which increased with the size of the alkyl group on the DNA. This could be attributed to the varied repair efficiencies of these lesions by O 6-alkylguanine DNA alkyltransferase (MGMT) in cells, which involve the MGMT Ogt and, to a lesser extent, Ada. In conclusion, our study provides important new knowledge about the repair of the O 6-alkyl-dG lesions and their recognition by the E. coli DNA replication machinery. Our results suggest that the lesions' carcinogenic potentials may be attributed, at least in part, to their strong mutagenic potential and their efficient bypass by the DNA replication machinery.
| S-EPMC6166734 | biostudies-literature
Project description:DNA alkylation represents a major type of DNA damage and is generally unavoidable due to ubiquitous exposure to various exogenous and endogenous sources of alkylating agents. Among the alkylated DNA lesions, O2-alkylthymidines (O2-alkyldT) are known to be persistent and poorly repaired in mammalian systems and have been shown to accumulate in the esophagus, lung, and liver tissue of rats treated with tobacco-specific N-nitrosamines, i.e., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). In this study, we assessed the replicative bypass of a comprehensive set of O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in template DNA by conducting primer extension assays with the use of major translesion synthesis DNA polymerases. The results showed that human Pol ? and, to a lesser degree, human Pol ?, but not human polymerase ? or yeast polymerase ?, were capable of bypassing all O2-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that human Pol ? exhibited high frequencies of misincorporation of dCMP opposite those O2-alkyldT lesions bearing a longer straight-chain alkyl group. However, the nucleotide misincorporation opposite branched-chain lesions was not selective, with dCMP, dGMP, and dTMP being inserted at similar efficiencies, though the total frequencies of nucleotide misincorporation opposite the branched-chain lesions differed and followed the order of O2-iPrdT > O2-iBudT > O2-sBudT. Together, the results from the present study provided important knowledge about the effects of the length and structure of the alkyl group in the O2-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol ?.
| S-EPMC5067237 | biostudies-literature
Project description:The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms. In Saccharomyces cerevisiae, dNTP concentration increases approximately 6- to 8-fold in response to DNA damage. This concentration increase is associated with improved tolerance of DNA damage, suggesting that translesion DNA synthesis is more efficient at elevated dNTP concentration. Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage. In vitro, under single-hit conditions, the replicative DNA polymerase epsilon does not bypass 7,8-dihydro-8-oxoguanine lesion (8-oxoG, one of the lesions produced by 4-NQO) at S-phase dNTP concentration, but does bypass the same lesion with 19-27% efficiency at DNA-damage-state dNTP concentration. The nucleotide inserted opposite 8-oxoG is dATP. We propose that during DNA damage in S. cerevisiae increased dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions.
| S-EPMC2553575 | biostudies-literature
Project description:N-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can also be generated endogenously. Metabolic activation of some NOCs can give rise to carboxymethylation and pyridyloxobutylation/pyridylhydroxybutylation of DNA, which are known to be carcinogenic and can lead to gastrointestinal and lung cancer, respectively. Herein, using the competitive replication and adduct bypass (CRAB) assay, along with MS- and NMR-based approaches, we assessed the cytotoxic and mutagenic properties of three O 6-alkyl-2'-deoxyguanosine (O 6-alkyl-dG) adducts, i.e. O 6-pyridyloxobutyl-dG (O 6-POB-dG) and O 6-pyridylhydroxybutyl-dG (O 6-PHB-dG), derived from tobacco-specific nitrosamines, and O 6-carboxymethyl-dG (O 6-CM-dG), induced by endogenous N-nitroso compounds. We also investigated two neutral analogs of O 6-CM-dG, i.e. O 6-aminocarbonylmethyl-dG (O 6-ACM-dG) and O 6-hydroxyethyl-dG (O 6-HOEt-dG). We found that, in Escherichia coli cells, these lesions mildly (O 6-POB-dG), moderately (O 6-PHB-dG), or strongly (O 6-CM-dG, O 6-ACM-dG, and O 6-HOEt-dG) impede DNA replication. The strong blockage effects of the last three lesions were attributable to the presence of hydrogen-bonding donor(s) located on the alkyl functionality of these lesions. Except for O 6-POB-dG, which also induced a low frequency of G → T transversions, all other lesions exclusively stimulated G → A transitions. SOS-induced DNA polymerases played redundant roles in bypassing all the O 6-alkyl-dG lesions investigated. DNA polymerase IV (Pol IV) and Pol V, however, were uniquely required for inducing the G → A transition for O 6-CM-dG exposure. Together, our study expands our knowledge about the recognition of important NOC-derived O 6-alkyl-dG lesions by the E. coli DNA replication machinery.
| S-EPMC6422096 | biostudies-literature
Project description:In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact.
| S-EPMC3660833 | biostudies-literature
Project description:SUMMARY:Replicative DNA polymerases are essential for the replication of the genomes of all living organisms. On the basis of sequence similarities they can be classified into three types. Type A polymerases are homologous to bacterial polymerases I, Type B comprises archaebacterial DNA polymerases and eukaryotic DNA polymerase alpha, and the bacterial polymerase III class make up type C. Structures have been solved for several type A and B polymerases, which share a similar architecture. The structure of type C is not yet known. The catalytic mechanism of all three types involves two metal-ion-binding acidic residues in the active site. Replicative polymerases are constitutively expressed, but their activity is regulated through the cell cycle and in response to different growth conditions.
| S-EPMC150442 | biostudies-literature
Project description:Genomic integrity is constantly challenged by a variety of endogenous and exogenous DNA damaging agents, which can lead to the formation of 104-105 DNA lesions per cell per day. Reactive oxygen species (ROS) represent a major type of DNA damaging agent. Specifically, a hydroxyl radical can attack the C1' position of 2-deoxyribose, and the ensuing carbon-centered radical, if improperly repaired, can cause the inversion of stereochemical configuration at the C1' to give ?-anomeric lesions. In this study, we assessed the replicative bypass of ?-dA, ?-dT, ?-dC, and ?-dG in template DNA by conducting primer extension assays with the use of purified translesion synthesis DNA polymerases. Our results revealed that human polymerase (Pol) ?, but not human Pol ?, Pol ?, or yeast Pol ?, was capable of bypassing all of the ?-dN lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol ? was the most efficient in inserting the correct nucleotides opposite the modified nucleosides, with the relative efficiencies of nucleotide incorporation following the order of ?-dA > ?-dG > ?-dT > ?-dC. Additionally, human Pol ? was found to misincorporate dTMP opposite ?-dT and dCMP opposite ?-dC at frequencies of 66% and 24%, respectively, whereas ?-dA and ?-dG were weakly miscoding. These findings provided important knowledge about the effects these ?-dN lesions have on the fidelity and efficiency of DNA replication mediated by human Pol ?.
| S-EPMC5614518 | biostudies-literature
Project description:One of the most common lesions induced by oxidative DNA damage is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Replicative DNA polymerases poorly traverse this highly mutagenic lesion, suggesting that the replication fork may switch to a polymerase specialized for translesion DNA synthesis (TLS) to catalyze 8-oxodG bypass in vivo. Here, we systematically compared the 8-oxodG bypass efficiencies and fidelities of the TLS-specialized, human Y-family DNA polymerases eta (hPol?), iota (hPol?), kappa (hPol?), and Rev1 (hRev1) either alone or in combination. Primer extension assays revealed that the times required for hPol?, hRev1, hPol?, and hPol? to bypass 50% of the 8-oxodG lesions encountered (t50(bypass)) were 0.58, 0.86, 108, and 670 s, respectively. Although hRev1 bypassed 8-oxodG efficiently, hRev1 failed to catalyze the extension step of TLS, and a second polymerase was required to extend the lesion bypass products. A high-throughput short oligonucleotide sequencing assay (HT-SOSA) was used to quantify the types and frequencies of incorporation errors produced by the human Y-family DNA polymerases at and near the 8-oxodG site. Although hPol? bypassed 8-oxodG most efficiently, hPol? correctly incorporated dCTP opposite 8-oxodG within only 54.5% of the sequences analyzed. In contrast, hPol? bypassed the lesion least efficiently but correctly incorporated dCTP at a frequency of 65.8% opposite the lesion. The combination of hRev1 and hPol? was most accurate opposite 8-oxodG (92.3%), whereas hPol? alone was the least accurate (18.5%). The t50(bypass) value and correct dCTP incorporation frequency in the presence of an equal molar concentration of all four Y-family enzymes were 0.60 s and 43.5%, respectively. These values are most similar to those of hPol? alone, suggesting that hPol? outcompetes the other three Y-family polymerases to catalyze 8-oxodG bypass in vitro and possibly in vivo.
| S-EPMC4033635 | biostudies-literature
Project description:Endogenous metabolites and exogenous chemicals can induce covalent modifications on DNA, producing DNA lesions. The N2 of guanine was shown to be a common alkylation site in DNA; however, not much is known about the influence of the size of the alkyl group in N2-alkyldG lesions on cellular DNA replication or how translesion synthesis (TLS) polymerases modulate DNA replication past these lesions in human cells. To answer these questions, we employ a robust shuttle vector method to investigate the impact of four N2-alkyldG lesions (i.e., with the alkyl group being a methyl, ethyl, n-propyl, or n-butyl group) on DNA replication in human cells. We find that replication through the N2-alkyldG lesions was highly efficient and accurate in HEK293T cells or isogenic CRISPR-engineered cells with deficiency in polymerase (Pol) ? or Pol ?. Genetic ablation of Pol ?, Pol ?, or Rev1, however, results in decreased bypass efficiencies and elicits substantial frequencies of G ? A transition and G ? T transversion mutations for these lesions. Moreover, further depletion of Pol ? in Pol ?- or Pol ?-deficient cells gives rise to elevated rates of G ? A and G ? T mutations and substantially decreased bypass efficiencies. Cumulatively, we demonstrate that the error-free replication past the N2-alkyldG lesions is facilitated by a specific subset of TLS polymerases, and we find that longer alkyl chains in these lesions induce diminished bypass efficiency and fidelity in DNA replication.
| S-EPMC6948715 | biostudies-literature