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Trinucleotide repeat (TNR) expansion is the cause of over 40 neurodegenerative diseases, including Huntington's disease and Friedreich's ataxia (FRDA). There are no effective treatments for these diseases due to the poor understanding of molecular mechanisms underlying somatic TNR expansion and contraction in neural systems. We and others have found that DNA base excision repair (BER) actively modulates TNR instability, shedding light on the development of effective treatments for the diseases by contracting expanded repeats through DNA repair. In this study, temozolomide (TMZ) was employed as a model DNA base damaging agent to reveal the mechanisms of the BER pathway in modulating GAA repeat instability at the frataxin (FXN) gene in FRDA neural cells and transgenic mouse mice. We found that TMZ induced large GAA repeat contraction in FRDA mouse brain tissue, neurons, and FRDA iPSC-differentiated neural cells, increasing frataxin protein levels in FRDA mouse brain and neural cells. Surprisingly, we found that TMZ could also inhibit H3K9 methyltransferases, leading to open chromatin and increasing ssDNA breaks and recruitment of the key BER enzyme, pol β, on the repeats in FRDA neural cells. We further demonstrated that the H3K9 methyltransferase inhibitor BIX01294 also induced the contraction of the expanded repeats and increased frataxin protein in FRDA neural cells by opening the chromatin and increasing the endogenous ssDNA breaks and recruitment of pol β on the repeats. Our study provides new mechanistic insight illustrating that inhibition of H3K9 methylation can crosstalk with BER to induce GAA repeat contraction in FRDA. Our results will open a new avenue for developing novel gene therapy by targeting histone methylation and the BER pathway for repeat expansion diseases. [ABSTRACT FROM AUTHOR]

Studies in knockout mice provide evidence that MSH2-MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2-MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2-MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion.

ABSTRACT Arsenite has been shown to induce a variety of oxidative damage in mammalian cells. However, the mechanisms underlying cellular responses to its adverse effects remain unknown. We previously showed that the level of Nrf2, a nuclear transcription factor significantly increased in arsenite-treated human bronchial epithelial (HBE) cells suggesting that Nrf2 is involved in responding to arsenite-induced oxidative damage. To explore how Nrf2 can impact arsenite-induced oxidative damage, in this study, we examined Nrf2 activation and its regulation upon cellular arsenite exposure as well as its effects on arsenite-induced oxidative damage in HBE cells. We found that Nrf2 mRNA and protein levels were significantly increased by arsenite in a dose- and time-dependent manner. Furthermore, we showed that over-expression of Nrf2 significantly reduced the level of arsenite-induced oxidative damage in HBE cells including DNA damage, chromosomal breakage, lipid peroxidation and depletion of antioxidants. This indicates a protective role of Nrf2 against arsenite toxicity. This was further supported by the fact that activation of Nrf2 by its agonists, tertiary butylhydroquinone (t-BHQ) and sulforaphane (SFN) resulted in the same protective effects against arsenite toxicity. Moreover, we demonstrated that arsenite-induced activation of Nrf2 was mediated by the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathway. This is the first evidence showing that Nrf2 protects against arsenite-induced oxidative damage through the cGMP-PKG pathway. Our study suggests that activation of Nrf2 through the cGMP-PKG signaling pathway in HBE cells may be developed as a new strategy for prevention of arsenite toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 2004-2020, 2017. [ABSTRACT FROM AUTHOR]

N 6 -methyladenosine (m 6 A), the most abundant and reversible RNA modification, plays critical a role in tumorigenesis. However, whether m 6 A can regulate p53, a leading antitumor protein remains poorly understood. In this study, we explored the regulatory role of m 6 A on p53 activation using an arsenite-transformed keratinocyte model, the HaCaT-T cell line. We created the cell line by exposing human keratinocyte HaCaT cells to 1 μM arsenite for 5 months. We found that the cells exhibited an increased m 6 A level along with an aberrant expression of the methyltransferases, demethylase, and readers of m 6 A. Moreover, the cells exhibited decreased p53 activity and reduced p53 phosphorylation, acetylation, and transactivation with a high nucleus export rate of p53. Knockdown of the m 6 A methyltransferase, METTL3 significantly decreased m 6 A level, restoring p53 activation and inhibiting cellular transformation phenotypes in the arsenite-transformed cells. Further, using both a bioinformatics analysis and experimental approaches, we demonstrated that m 6 A downregulated the expression of the positive p53 regulator, PRDM2, through the YTHDF2-promoted decay of PRDM2 mRNAs. We showed that m 6 A upregulated the expression of the negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. Taken together, our study revealed the novel role of m 6 A in mediating arsenite-induced human keratinocyte transformation by suppressing p53 activation. This study further sheds light on the mechanisms of arsenic carcinogenesis via RNA epigenetics., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Trinucleotide repeat (TNR) instability (expansion and deletion) is associated with more than 42 human neurodegenerative diseases and cancer and mediated by DNA replication, repair, recombination, and gene transcription. Somatic TNR instability is involved in the progression of TNR expansion diseases and can be modulated by DNA damage repair and gene transcription. Recent studies from our group and others have shown that DNA base damage and its repair play an active role in modulating TNR instability and are responsible for somatic age-dependent CAG repeat expansion in neurons of Huntington's disease mice induced by oxidative DNA damage. However, it remains to be elucidated how DNA damage, non-B form DNA structures, and DNA repair enzymes and cofactors can coordinate to regulate somatic TNR instability. Understanding the molecular mechanisms underlying DNA damage and repair-mediated somatic TNR instability is critically important for identification of new therapeutic targets for treatment and prevention of TNR-related diseases. Here we describe the methods to study the locations and distribution of DNA base lesions and their effects on TNR instability through DNA base excision repair in in vitro reconstituted human systems.

Objective: To explore the effect of DNA polymerase beta (pol beta) expression level on genotoxicity in mouse fibroblast cell induced by hydroquinone (HQ)., Methods: pol beta wild-type cells (pol beta +/+), pol beta null cells (pol beta -/-) and pol beta overexpressed cells (pol beta oe) were applied as a model cell system. At various concentration of HQ, the cell cytotoxicities were detected by MTT assay. The DCFH-DA fluorescent probe was used to examine the cellular ROS levels. The effects of DNA damage/repair and chromosomal damage were observed by comet assay and micronuleus assay respectively., Results: MTT assay showed that the doses of HQ increased, the cells viabilities were decreased. The concentrations of HQ that inhibited cell growth by 50% (IC50) in the pol beta -/- cell were more lower than those of other cells (P < 0.05). The cellular ROS level in the three kinds of cells were increased in a concentration dependent way after treated with HQ and it was more stronger in pol beta -/- cell than those in other two kinds of cells (P < 0.05). Comet assay and micronucleus assay showed that HQ induced DNA damage and increased micronucleus formation. DNA and chromosomal damage of pol beta -/- cell were more serious than those of other cells. The DNA damage repair capacities of pol beta oe cell were more stronger than pol beta +/+ and pol beta -/- cells., Conclusion: Cellular ROS generation could be effectively induced by HQ, leading to DNA and chromosomal damage, pol beta overexpression could help cells response to oxidative damage and protect cells from genotoxicity induced by HQ.

Objective: To clarify the relationship between the expression of pol beta and DNA damage /repair induced by Benzo(a)pyrene (BaP)., Methods: pol beta wild-type cells (pol beta +/+), pol beta null cells (pol beta -/-) and wild-type pol beta overexpressed cells (pol beta oe) which had the same genetic background were studied. Firstly, RT-PCR and Western blot targeting to pol beta were carried out to measure the expression of pol beta mRNA and protein in above three kinds of cells, then MTT test and single cell gel electrophoresis (comet assay) were used to compare cell viability and DNA damage/repair of the three kinds of cells when exposed to BaP., Results: There was pol beta deletion in pol beta -/- cells and the level of pol beta mRNA and protein in pol beta oe cells was twice higher than that in pol beta +/+ cells. BaP could induce DNA damage and reduce cell viability, when compared with pol beta +/+ cells, IC50 of pol beta -/- cells was remarkably lower, DNA was prone to damage and more difficult to be repaired, on the other hand, IC50 of pol beta oe cells was obviously higher and the damage effect on DNA was weaker and prone to be repaired., Conclusion: Pol beta played an important role in the repair of DNA damage induced by BaP, deficiency of pol beta could decrease the DNA repair capability of cells, and overexpression of pol beta could help cells response to DNA damage and protect cells from death in a certain degree.