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Mitochondrial Transcription Factor A Binds to and Promotes Mutagenic Transcriptional Bypass of O⁴-Alkylthymidine Lesions.

ABSTRACT: O²- and O⁴-alkylated thymidine lesions are known to be poorly repaired and persist in mammalian tissues. To understand how mammalian cells sense the presence and regulate the repair of these lesions, we employed a quantitative proteomic method to discover regioisomeric O²- and O⁴-n-butylthymidine (O²- and O⁴-nBudT)-binding proteins. We were able to identify 21 and 74 candidate DNA damage recognition proteins for O²-nBudT- and O⁴-nBudT-bearing DNA probes, respectively. Among these proteins, DDB1 and DDB2 selectively bind to O²-nBudT-containing DNA, whereas three high-mobility group (HMG) proteins (i.e., HMGB1, HMGB2, and mitochondrial transcription factor A (TFAM)) exhibit preferential binding to O⁴-nBudT-bearing DNA. We further demonstrated that TFAM binds directly and selectively with O⁴-alkyldT-harboring DNA, and the binding capacity depends mainly on the HMG box-A domain of TFAM. We also found that TFAM promotes transcriptional mutagenesis of O⁴-nBudT and O⁴-pyridyloxobutylthymidine, which is a DNA adduct induced by tobacco-specific N-nitrosamines, in vitro and in human cells. Together, we explored, for the first time, the interaction proteomes of O-alkyldT lesions, and our study expanded the functions of TFAM by revealing its capability in the recognition of O⁴-alkyldT-bearing DNA and uncovering its modulation of transcriptional mutagenesis of these lesions in human cells.

SUBMITTER: He X

PROVIDER: S-EPMC7904241 | biostudies-literature |

REPOSITORIES: biostudies-literature

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TFAM Binds to and Promotes Mutagenic Transcriptional Bypass of O4-Alkylthymidine Lesions

Project description:O2- and O4-alkylated thymidine lesions are known to be poorly repaired and persist in mammalian tissues. To understand how mammalian cells sense the presence and regulate the repair of these lesions, we employed a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic method to discover novel O2- and O4-n-butylthymidine (O2- and O4-nBudT)-binding proteins. We were able to identify 21 and 74 candidate DNA damage recognition proteins for O2-nBudT- and O4-nBudT-bearing DNA probes, respectively. Among these proteins, DDB1 and DDB2 selectively bind to O2-nBudT-containing DNA, whereas three HMG-box-containing proteins (i.e. HMGB1, HMGB2 and TFAM) exhibit preferential binding to O4-nBudT-bearing DNA. We further confirmed, by employing electrophoretic mobility shift assay, that TFAM can bind selectively and directly with O4-alkyldT-harboring DNA, and the binding capacity depends mainly on the HMG box-A domain of TFAM. We also found that TFAM promotes transcriptional mutagenesis of O4-alkyldT lesions in vitro and in human cells. Together, we explored, for the first time, the interactomes of O-alkyldT lesions. Our study also expands the functions of TFAM by revealing its capability in binding to O4-alkyldT-bearing DNA, demonstrating the role of HMG-box A domain in this molecular recognition, and uncovering its modulation of transcriptional mutagenesis of these lesions in human cells.

2020-12-21 | PXD021719 | Pride

Cytotoxic and mutagenic properties of minor-groove O2-alkylthymidine lesions in human cells.

Project description:Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, are recognized by the DNA replication machinery in human cells. We found that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T→A and T→G mutations. Replication experiments using isogenic cells deficient in specific translesion synthesis (TLS) DNA polymerases revealed that the absence of polymerase η or polymerase ζ, but not polymerase κ or polymerase ι, significantly decreased both the bypass efficiencies and the mutation frequencies for those O2-alkyldT lesions carrying a straight-chain alkyl group. Moreover, the mutagenic properties of the O2-alkyldT lesions were influenced by the length and topology of the alkyl chain and by TLS polymerases. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of O2-alkyldT lesions, and illustrate the roles of TLS polymerases in replicative bypass of these lesions in human cells.

| S-EPMC5986211 | biostudies-literature

Replicative Bypass of O2-Alkylthymidine Lesions in Vitro.

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

Syntheses and characterizations of the in vivo replicative bypass and mutagenic properties of the minor-groove O2-alkylthymidine lesions.

Project description:Endogenous metabolism, environmental exposure, and treatment with some chemotherapeutic agents can all give rise to DNA alkylation, which can occur on the phosphate backbone as well as the ring nitrogen or exocyclic nitrogen and oxygen atoms of nucleobases. Previous studies showed that the minor-groove O(2)-alkylated thymidine (O(2)-alkyldT) lesions are poorly repaired and persist in mammalian tissues. In the present study, we synthesized oligodeoxyribonucleotides harboring seven O(2)-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, at a defined site and examined the impact of these lesions on DNA replication in Escherichia coli cells. Our results demonstrated that the replication bypass efficiencies of the O(2)-alkyldT lesions decreased with the chain length of the alkyl group, and these lesions directed promiscuous nucleotide misincorporation in E. coli cells. We also found that deficiency in Pol V, but not Pol II or Pol IV, led to a marked drop in bypass efficiencies for most O(2)-alkyldT lesions. We further showed that both Pol IV and Pol V were essential for the misincorporation of dCMP opposite these minor-groove DNA lesions, whereas only Pol V was indispensable for the T?A transversion introduced by these lesions. Depletion of Pol II, however, did not lead to any detectable alterations in mutation frequencies for any of the O(2)-alkyldT lesions. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O(2)-alkyldT lesions and revealed the roles of the SOS-induced DNA polymerases in bypassing these lesions in E. coli cells.

| S-EPMC4176383 | biostudies-literature

Cytotoxic and mutagenic properties of O4-alkylthymidine lesions in Escherichia coli cells.

Project description:Due to the abundant presence of alkylating agents in living cells and the environment, DNA alkylation is generally unavoidable. Among the alkylated DNA lesions, O(4)-alkylthymidine (O(4)-alkyldT) are known to be highly mutagenic and persistent in mammalian tissues. Not much is known about how the structures of the alkyl group affect the repair and replicative bypass of the O(4)-alkyldT lesions, or how the latter process is modulated by translesion synthesis polymerases. Herein, we synthesized oligodeoxyribonucleotides harboring eight site-specifically inserted O(4)-alkyldT lesions and examined their impact on DNA replication in Escherichia coli cells. We showed that the replication past all the O(4)-alkyldT lesions except (S)- and (R)-sBudT was highly efficient, and these lesions directed very high frequencies of dGMP misincorporation in E. coli cells. While SOS-induced DNA polymerases play redundant roles in bypassing most of the O(4)-alkyldT lesions, the bypass of (S)- and (R)-sBudT necessitated Pol V. Moreover, Ada was not involved in the repair of any O(4)-alkyldT lesions, Ogt was able to repair O(4)-MedT and, to a lesser extent, O(4)-EtdT and O(4)-nPrdT, but not other O(4)-alkyldT lesions. Together, our study provided important new knowledge about the repair of the O(4)-alkyldT lesions and their recognition by the E. coli replication machinery.

| S-EPMC4678858 | biostudies-literature

EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions.

Project description:The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress; however, the damage tolerance of these key cellular functions comes at the expense of fidelity. Thus, translesion DNA synthesis (TLS) over damaged nucleotides is a major source of point mutations found in cancers; whereas erroneous bypass of damage by RNA polymerases may contribute to cancer and other diseases by driving accumulation of proteins with aberrant structure and function in a process termed "transcriptional mutagenesis" (TM). Here, we aimed at the generation of reporters suited for direct detection of miscoding capacities of defined types of DNA modifications during translesion DNA or RNA synthesis in human cells. We performed a systematic phenotypic screen of 25 non-synonymous base substitutions in a DNA sequence encoding a functionally important region of the enhanced green fluorescent protein (EGFP). This led to the identification of four loss-of-fluorescence mutants, in which any ulterior base substitution at the nucleotide affected by the primary mutation leads to the reversal to a functional EGFP. Finally, we incorporated highly mutagenic abasic DNA lesions at the positions of primary mutations and demonstrated a high sensitivity of detection of the mutagenic DNA TLS and TM in this system.

| S-EPMC7357151 | biostudies-literature

Implications for damage recognition during Dpo4-mediated mutagenic bypass of m1G and m3C lesions.

Project description:DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3'-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.

| S-EPMC3744880 | biostudies-literature

Translesion synthesis of O4-alkylthymidine lesions in human cells.

Project description:Environmental exposure, endogenous metabolism and cancer chemotherapy can give rise to alkylation of DNA, and the resulting alkylated thymidine (alkyldT) lesions were found to be poorly repaired and persistent in mammalian tissues. Unrepaired DNA lesions may compromise genomic integrity by inhibiting DNA replication and inducing mutations in these processes. In this study, we explored how eight O4-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, (R)-sBu and (S)-sBu, are recognized by DNA replication machinery in HEK293T human embryonic kidney cells. We found that the O4-alkyldT lesions are moderately blocking to DNA replication, with the bypass efficiencies ranging from 20 to 33% in HEK293T cells, and these lesions induced substantial frequencies T?C transition mutation. We also conducted the replication experiments in the isogenic cells where individual translesion synthesis (TLS) DNA polymerases were depleted by the CRISPR/Cas9 genome editing method. Our results showed that deficiency in Pol ? or Pol ?, but not Pol ? or Pol ?, led to pronounced drops in bypass efficiencies for all the O4-alkyldT lesions except O4-MedT. In addition, depletion of Pol ? resulted in significant decreases in T?C mutation frequencies for all the O4-alkyldT lesions except O4-MedT and O4-nBudT. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O4-alkyldT lesions and defined the roles of TLS polymerases in bypassing these lesions in human cells.

| S-EPMC5100597 | biostudies-literature

Transcription bypass of DNA lesions enhances cell survival but attenuates transcription coupled DNA repair.

Project description:Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) dedicated to rapid removal of DNA lesions in the transcribed strand of actively transcribed genes. The precise nature of the TCR signal and how the repair machinery gains access to lesions imbedded in stalled RNA polymerase II (RNAP II) complexes in eukaryotic cells are still enigmatic. RNAP II has an intrinsic capacity for transcription bypass of DNA lesions by incorporation or misincorporation of nucleotides across the lesions. It has been suggested that transcription bypass of lesions, which exposes the lesions, may be required for TCR. Here, we show that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced cyclobutane pyrimidine dimers (CPDs), increases survival of UV irradiated yeast cells but attenuates TCR. The increased cell survival is independent of any NER subpathways. In contrast, G730D mutation of Rpb1, which impairs transcription bypass of CPDs, enhances TCR. Our results suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR.

| S-EPMC4245964 | biostudies-literature

Cytotoxic and mutagenic properties of O 6-alkyl-2'-deoxyguanosine lesions in Escherichia coli cells.

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