Translesion synthesis of<i>O</i><sup>4</sup>-alkylthymidine lesions in human cells

Wu, Jun; Li, Lin; Wang, Pengcheng; You, Changjun; Williams, Nicole L.; Wang, Yinsheng

doi:10.1093/nar/gkw662

Cited by 46 publications

(124 citation statements)

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“…The complete and selective depletion of individual TLS polymerases was confirmed previously by DNA sequencing and Western blot analyses (23). …”

Section: Methodssupporting

confidence: 60%

Replication studies of carboxymethylated DNA lesions in human cells

Wang

et al. 2017

Nucleic Acids Research

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Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.e. O6-CMdG, N6-CMdA, N4-CMdC, N3-CMdT and O4-CMdT on the efficiency and fidelity of DNA replication in HEK293T human embryonic kidney cells. We found that, while neither N6-CMdA nor N4-CMdC blocked DNA replication or induced mutations, N3-CMdT, O4-CMdT and O6-CMdG moderately blocked DNA replication and induced substantial frequencies of T→A (81%), T→C (68%) and G→A (6.4%) mutations, respectively. In addition, our results revealed that CRISPR-Cas9-mediated depletion of Pol η resulted in significant drops in bypass efficiencies of N4-CMdC and N3-CMdT. Diminution in bypass efficiencies was also observed for N6-CMdA and O6-CMdG upon depletion of Pol κ, and for O6-CMdG upon removal of Pol ζ. Together, our study provided molecular-level insights into the impacts of the carboxymethylated DNA lesions on DNA replication in human cells, revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions, and suggested the contributions of O6-CMdG, N3-CMdT and O4-CMdT to the mutations found in p53 gene of human gastrointestinal cancers.

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“…The complete and selective depletion of individual TLS polymerases was confirmed previously by DNA sequencing and Western blot analyses (23). …”

Section: Methodssupporting

confidence: 60%

Replication studies of carboxymethylated DNA lesions in human cells

Wang

et al. 2017

Nucleic Acids Research

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“…36 Additionally, human Pol η is capable of promoting replication through many other DNA lesions including O 6 -alkyldG, 8-oxodG, O 4 -alkyldT, O 2 -MedT and O 2 -POBdT. 30, 36, 41, 42 This may explain why human Pol η is able to incorporate a nucleotide opposite all seven O 2 -alkyldT lesions and extend past them. Moreover, the high frequency of misincorporation of dCMP opposite the longer straight-chain lesions, i.e.…”

Section: Discussionmentioning

confidence: 99%

“…30 Additionally, human Pol η and ζ were found to promote the bypass of the O 4 -alkyldT lesions in HEK293T cells. 42 On the other hand, the nature of nucleotide misincorporation mediated by the polymerase is vastly different when bypassing the dT lesions with the same alkyl groups being attached to the O 2 and O 4 positions. Unlike our current findings where dCMP was the main misincorporation event identified for the minor-groove O 2 -alkyldT lesions, dGMP was found to be the major nucleotide misincorporated opposite the major-groove O 4 -alkyldT lesions both in vitro and in cells.…”

Section: Discussionmentioning

confidence: 99%

Replicative Bypass of O²-Alkylthymidine Lesions in Vitro

Williams

Wang

2016

Chem. Res. Toxicol.

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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 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 η and, to a lesser degree, human Pol κ, but not human polymerase ι or yeast polymerase ζ, 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 η 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 η.

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“…To test the hypothesis that hpol is the major reverse transcriptase in other human cells, extracts from HEK293T cells were also examined. TLS DNA polymerase-deficient cell lines, deficient in hpols , , , and , respectively, were generated and extracts were prepared (49). These cell extracts were capable of extending a DNA primer opposite an undamaged DNA template, although significant degradation was observed ( Fig.…”

Section: Hpol Is the Major Reverse Transcriptase In Hek293t Cellsmentioning

confidence: 99%

Human DNA polymerase η has reverse transcriptase activity in cellular environments

Ghodke

Egli

et al. 2019

Journal of Biological Chemistry

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Edited by Patrick SungClassical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol (hpol ) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol , could not add rNTPs to a DNA primer, but the expression of transfected hpol in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol , , or -were individually deleted. These results suggest that hpol is one of the major reverse transcriptases involved in physiological processes in human cells.

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Translesion synthesis ofO⁴-alkylthymidine lesions in human cells

Cited by 46 publications

References 40 publications

Replication studies of carboxymethylated DNA lesions in human cells

Replication studies of carboxymethylated DNA lesions in human cells

Replicative Bypass of O²-Alkylthymidine Lesions in Vitro

Human DNA polymerase η has reverse transcriptase activity in cellular environments

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Translesion synthesis ofO4-alkylthymidine lesions in human cells

Cited by 46 publications

References 40 publications

Replication studies of carboxymethylated DNA lesions in human cells

Replication studies of carboxymethylated DNA lesions in human cells

Replicative Bypass of O2-Alkylthymidine Lesions in Vitro

Human DNA polymerase η has reverse transcriptase activity in cellular environments

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Translesion synthesis ofO⁴-alkylthymidine lesions in human cells

Replicative Bypass of O²-Alkylthymidine Lesions in Vitro