Journal articles on the topic 'In vitro DNA Repair Mechanisms'
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1
Dikomey, E. "Bestimmung der DNA-Schädigung in vitro." Nuklearmedizin 49, S 01 (2010): S64—S68. http://dx.doi.org/10.1055/s-0038-1626526.
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SummaryIonising irradiation acts primarily via induction of DNA damage, among which doublestrand breaks are the most important lesions. These lesions may lead to lethal chromosome aberrations, which are the main reason for cell inactivation. Double-strand breaks can be repaired by several different mechanisms. The regulation of these mechanisms appears be fairly different for normal and tumour cells. Among different cell lines capacity of doublestrand break repair varies by only few percents and is known to be determined mostly by genetic factors. Knowledge about doublestrand break repair mechanisms and their regulation is important for the optimal application of ionising irradiation in medicine.
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Guo, Hongrui, Huan Liu, Hongbin Wu, Hengmin Cui, Jing Fang, Zhicai Zuo, Junliang Deng, Yinglun Li, Xun Wang, and Ling Zhao. "Nickel Carcinogenesis Mechanism: DNA Damage." International Journal of Molecular Sciences 20, no. 19 (September 21, 2019): 4690. http://dx.doi.org/10.3390/ijms20194690.
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Nickel (Ni) is known to be a major carcinogenic heavy metal. Occupational and environmental exposure to Ni has been implicated in human lung and nasal cancers. Currently, the molecular mechanisms of Ni carcinogenicity remain unclear, but studies have shown that Ni-caused DNA damage is an important carcinogenic mechanism. Therefore, we conducted a literature search of DNA damage associated with Ni exposure and summarized known Ni-caused DNA damage effects. In vitro and vivo studies demonstrated that Ni can induce DNA damage through direct DNA binding and reactive oxygen species (ROS) stimulation. Ni can also repress the DNA damage repair systems, including direct reversal, nucleotide repair (NER), base excision repair (BER), mismatch repair (MMR), homologous-recombination repair (HR), and nonhomologous end-joining (NHEJ) repair pathways. The repression of DNA repair is through direct enzyme inhibition and the downregulation of DNA repair molecule expression. Up to now, the exact mechanisms of DNA damage caused by Ni and Ni compounds remain unclear. Revealing the mechanisms of DNA damage from Ni exposure may contribute to the development of preventive strategies in Ni carcinogenicity.
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Lai, Ying-Ta, and Warren Masker. "In Vitro Repair of Gaps in Bacteriophage T7 DNA." Journal of Bacteriology 180, no. 23 (December 1, 1998): 6193–202. http://dx.doi.org/10.1128/jb.180.23.6193-6202.1998.
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ABSTRACT An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.
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Freyer, G. A., S. Davey, J. V. Ferrer, A. M. Martin, D. Beach, and P. W. Doetsch. "An alternative eukaryotic DNA excision repair pathway." Molecular and Cellular Biology 15, no. 8 (August 1995): 4572–77. http://dx.doi.org/10.1128/mcb.15.8.4572.
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DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.
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Kunkel, Thomas A., Katarzyna Bebenek, John D. Roberts, Mary P. Fitzgerald, and David C. Thomas. "Analysis of fidelity mechanisms with eukaryotic DNA replication and repair proteins." Genome 31, no. 1 (January 1, 1989): 100–103. http://dx.doi.org/10.1139/g89-019.
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We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (α, β, δ, γ), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells.Key words: error frequency, repair proteins, polymerases, mutational specificity, fidelity mechanisms.
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George, Vazhappilly Cijo, and H. P. Vasantha Rupasinghe. "Apple Flavonoids Suppress Carcinogen-Induced DNA Damage in Normal Human Bronchial Epithelial Cells." Oxidative Medicine and Cellular Longevity 2017 (2017): 1–12. http://dx.doi.org/10.1155/2017/1767198.
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Scope. Human neoplastic transformation due to DNA damage poses an increasing global healthcare concern. Maintaining genomic integrity is crucial for avoiding tumor initiation and progression. The present study aimed to investigate the efficacy of an apple flavonoid fraction (AF4) against various carcinogen-induced toxicity in normal human bronchial epithelial cells and its mechanism of DNA damage response and repair processes. Methods and Results. AF4-pretreated cells were exposed to nicotine-derived nitrosamine ketones (NNK), NNK acetate (NNK-Ae), methotrexate (MTX), and cisplatin to validate cytotoxicity, total reactive oxygen species, intracellular antioxidants, DNA fragmentation, and DNA tail damage. Furthermore, phosphorylated histone (γ-H2AX) and proteins involved in DNA damage (ATM/ATR, Chk1, Chk2, and p53) and repair (DNA-PKcs and Ku80) mechanisms were evaluated by immunofluorescence and western blotting, respectively. The results revealed that AF4-pretreated cells showed lower cytotoxicity, total ROS generation, and DNA fragmentation along with consequent inhibition of DNA tail moment. An increased level of γ-H2AX and DNA damage proteins was observed in carcinogen-treated cells and that was significantly (p≤0.05) inhibited in AF4-pretreated cells, in an ATR-dependent manner. AF4 pretreatment also facilitated the phosphorylation of DNA-PKcs and thus initiation of repair mechanisms. Conclusion. Apple flavonoids can protect in vitro oxidative DNA damage and facilitate repair mechanisms.
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Tadi, Satish Kumar, Robin Sebastian, Sumedha Dahal, Ravi K. Babu, Bibha Choudhary, and Sathees C. Raghavan. "Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions." Molecular Biology of the Cell 27, no. 2 (January 15, 2016): 223–35. http://dx.doi.org/10.1091/mbc.e15-05-0260.
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Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.
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Kuzminov, Andrei. "Recombinational Repair of DNA Damage inEscherichia coli and Bacteriophage λ." Microbiology and Molecular Biology Reviews 63, no. 4 (December 1, 1999): 751–813. http://dx.doi.org/10.1128/mmbr.63.4.751-813.1999.
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SUMMARY Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.
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Kozhina, R. A., V. N. Chausov, E. A. Kuzmina, and A. V. Boreyko. "Induction and repair of DNA double-strand breaks in hippocampal neurons of miсe of different age after exposure to 60Со γ-rays in vivo and in vitro." EPJ Web of Conferences 177 (2018): 06001. http://dx.doi.org/10.1051/epjconf/201817706001.
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One of the central problems of modern radiobiology is the study of DNA damage induction and repair mechanisms in central nervous system cells, in particular, in hippocampal cells. The study of the regularities of molecular damage formation and repair in the hippocampus cells is of special interest, because these cells, unlike most cells of the central nervous system (CNS), keep proliferative activity, i.e. ability to neurogenesis. Age-related changes in hippocampus play an important role, which could lead to radiosensitivity changes in neurons to the ionizing radiation exposure. Regularities in DNA double-strand breaks (DSB) induction and repair in different aged mice hippocampal cells in vivo and in vitro under the action of γ-rays 60Со were studied with DNA comet-assay. The obtained dose dependences of DNA DSB induction are linear both in vivo and in vitro. It is established that in young animals' cells, the degree of DNA damage is higher than in older animals. It is shown that repair kinetics is basically different for exposure in vivo and in vitro.
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Lans, Hannes, and Wim Vermeulen. "Nucleotide Excision Repair in Caenorhabditis elegans." Molecular Biology International 2011 (August 17, 2011): 1–12. http://dx.doi.org/10.4061/2011/542795.
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Nucleotide excision repair (NER) plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR) mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will discuss current knowledge gained from the use of C. elegans to study NER and the response to UV-induced DNA damage.
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Lee, Shih-Yu, I.-Chuan Yen, Jang-Chun Lin, Min-Chieh Chung, and Wei-Hsiu Liu. "4-Acetylantrocamol LT3 Inhibits Glioblastoma Cell Growth and Downregulates DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase." American Journal of Chinese Medicine 49, no. 04 (January 2021): 983–99. http://dx.doi.org/10.1142/s0192415x21500476.
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Glioblastoma multiforme (GBM) is a deadly malignant brain tumor that is resistant to most clinical treatments. Novel therapeutic agents that are effective against GBM are required. Antrodia cinnamomea has shown antiproliferative effects in GBM cells. However, the exact mechanisms and bioactive components remain unclear. Thus, the present study aimed to investigate the effect and mechanism of 4-acetylantrocamol LT3 (4AALT3), a new ubiquinone from Antrodia cinnamomeamycelium, in vitro. U87 and U251 cell lines were treated with the indicated concentration of 4AALT3. Cell viability, cell colony-forming ability, migration, and the expression of proteins in well-known signaling pathways involved in the malignant properties of glioblastoma were then analyzed by CCK-8, colony formation, wound healing, and western blotting assays, respectively. We found that 4AALT3 significantly decreased cell viability, colony formation, and cell migration in both in vitro models. The epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), Hippo/yes-associated protein (YAP), and cAMP-response element binding protein (CREB) pathways were suppressed by 4AALT3. Moreover, 4AALT3 decreased the level of DNA repair enzyme O6-methylguanine-DNA methyltransferase and showed a synergistic effect with temozolomide. Our findings provide the basis for exploring the beneficial effect of 4AALT3 on GBM in vivo.
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Pavlova, Anzhela V., Elena A. Kubareva, Mayya V. Monakhova, Maria I. Zvereva, and Nina G. Dolinnaya. "Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair." Biomolecules 11, no. 9 (August 27, 2021): 1284. http://dx.doi.org/10.3390/biom11091284.
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DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here.
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Lohberger, Birgit, Dietmar Glänzer, Nicole Eck, Katharina Stasny, Anna Falkner, Andreas Leithner, and Dietmar Georg. "The ATR Inhibitor VE-821 Enhances the Radiosensitivity and Suppresses DNA Repair Mechanisms of Human Chondrosarcoma Cells." International Journal of Molecular Sciences 24, no. 3 (January 24, 2023): 2315. http://dx.doi.org/10.3390/ijms24032315.
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To overcome the resistance to radiotherapy in chondrosarcomas, the prevention of efficient DNA repair with an additional treatment was explored for particle beams as well as reference X-ray irradiation. The combined treatment with DNA repair inhibitors—with a focus on ATRi VE-821—and proton or carbon ions irradiation was investigated regarding cell viability, proliferation, cell cycle distribution, MAPK phosphorylation, and the expression of key DNA repair genes in two human chondrosarcoma cell lines. Pre-treatment with the PARPis Olaparib or Veliparib, the ATMi Ku-55933, and the ATRi VE-821 resulted in a dose-dependent reduction in viability, whereas VE-821 has the most efficient response. Quantification of γH2AX phosphorylation and protein expression of the DNA repair pathways showed a reduced regenerative capacity after irradiation. Furthermore, combined treatment with VE-821 and particle irradiation increased MAPK phosphorylation and the expression of apoptosis markers. At the gene expression and at the protein expression/phosphorylation level, we were able to demonstrate the preservation of DNA damage after combined treatment. The present data showed that the combined treatment with ATMi VE-821 increases the radiosensitivity of human chondrosarcoma cells in vitro and significantly suppresses efficient DNA repair mechanisms, thus improving the efficiency of radiotherapy.
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Aengenvoort, Jana, Marlena Sekeres, Peter Proksch, and Gerhard Fritz. "Targeting Mechanisms of the DNA Damage Response (DDR) and DNA Repair by Natural Compounds to Improve cAT-Triggered Tumor Cell Death." Molecules 27, no. 11 (June 1, 2022): 3567. http://dx.doi.org/10.3390/molecules27113567.
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Recently, we identified secalonic acid F (SA), 5-epi-nakijiquinone Q (NQ) and 5-epi-ilimaquinone (IQ) as natural compounds (NC) affecting mechanisms of the DNA damage response (DDR). Here, we further characterized their effects on DDR, DNA repair and cytotoxicity if used in mono- and co-treatment with conventional anticancer therapeutics (cAT) (cisplatin (Cis), doxorubicin (Doxo)) in vitro. All three NC influence the phosphorylation level of selected DDR-related factors (i.e., pCHK1, pKAP1, pP53, pRPA32) in mono- and/or co-treatment. Both SA and NQ attenuate the Cis- and Doxo-induced G2/M-phase arrest and effectively stimulate caspase-mediated apoptosis. Notably, SA impacts DNA repair as reflected by enhanced steady-state levels of Cis-(1,2-GpG)-DNA adducts and Doxo-induced DNA double-strand breaks (DSB). Moreover, SA decreased the mRNA and protein expression of the homologous recombination (HR)-related DSB repair factors RAD51 and BRCA1. Both SA and NQ promote Cis- and Doxo-induced cytotoxicity in an additive to synergistic manner (CI ≤ 1.0). Summarizing, we conclude that SA promotes cAT-driven caspase-dependent cell death by interfering with DSB repair and DDR-related checkpoint control mechanisms. Hence, SA is considered as the most promising lead compound to evaluate its therapeutic window in forthcoming pre-clinical in vivo studies.
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Stepanenko, Aleksei A., and Vladimir P. Chekhonin. "On the Critical Issues in Temozolomide Research in Glioblastoma: Clinically Relevant Concentrations and MGMT-independent Resistance." Biomedicines 7, no. 4 (November 27, 2019): 92. http://dx.doi.org/10.3390/biomedicines7040092.
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The current standard first-line treatment for adult patients with newly diagnosed glioblastoma includes concurrent radiotherapy and daily oral temozolomide (TMZ), followed by adjuvant TMZ. As a prodrug, TMZ undergoes spontaneous hydrolysis generating a methylating agent. O6-methylguanine is considered the most preponderant toxic damage mechanism at therapeutically relevant TMZ doses, whereas MGMT, which encodes the O6-methylguanine-DNA methyltransferase DNA repair enzyme, is the most relevant resistance mechanism. Speculations on clinically relevant TMZ concentrations, cytotoxic and cytostatic effects of TMZ, and resistance mechanisms exist in the literature. Here, we raise the following principal issues: What are the clinically relevant TMZ concentrations in glioma patients, and which TMZ-induced molecular lesion(s) and corresponding resistance mechanism(s) are important for TMZ therapeutic effects at clinically relevant concentrations? According to clinical data from patients with glioblastoma, the mean peak TMZ concentrations in the peritumoral tissue might be much lower (around 5 µM) than usually used in in vitro research, and may represent only 20% of systemic drug levels. According to in vitro reports, single-dose TMZ at concentrations around 5 µM have minimal, if any, effect on apoptosis and/or senescence of glioblastoma cell lines. However, the clinically relevant concentrations of TMZ are sufficient to radiosensitize both MGMT-positive and -negative cell lines in vitro. It is speculated that a single DNA repair protein, MGMT, is highly efficient in protecting cells against TMZ toxicity. However, an endogenous level of MGMT protein expression is not universally correlated with TMZ responsiveness, and MGMT-independent mechanisms of TMZ resistance exist.
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Barvaux, Vincent A., Malcolm Ranson, Robert Brown, R. Stanley McElhinney, T. Brian H. McMurry, and Geoffrey P. Margison. "Dual repair modulation reverses Temozolomide resistance in vitro." Molecular Cancer Therapeutics 3, no. 2 (February 1, 2004): 123–27. http://dx.doi.org/10.1158/1535-7163.123.3.2.
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Abstract Temozolomide is an alkylating agent that mediates its cytotoxic effects via O6-methylguanine (O6-meG) adducts in DNA and their recognition and processing by the postreplication mismatch repair system (MMR). O6-meG adducts can be repaired by the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (MGMT), which therefore constitutes a major resistance mechanism to the drug. Resistance to Temozolomide can also be mediated by loss of MMR, which is frequently mediated by methylation of the hMLH1 gene promoter. Methylation of hMLH1 can be reversed by treatment of cells with 5-aza-2′-deoxycytidine, while the MGMT pseudosubstrate O6-(4-bromothenyl)guanine (PaTrin-2) can deplete MGMT activity. Using a drug-resistant cell line which expresses MGMT and has methylated hMLH1, we show that while either of these treatments can individually sensitize cells to Temozolomide, the combined treatment leads to substantially greater sensitization. The increased sensitization is not observed in matched MMR proficient cells.
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Feng, Maoxiao, Yunshan Wang, Lei Bi, Pengju Zhang, Huaizhi Wang, Zhongxi Zhao, Jian-Hua Mao, and Guangwei Wei. "CRL4ADTL degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation." Oncogene 40, no. 11 (February 24, 2021): 2096–111. http://dx.doi.org/10.1038/s41388-021-01690-z.
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AbstractGenomic instability induced by DNA damage and improper DNA damage repair is one of the main causes of malignant transformation and tumorigenesis. DNA double strand breaks (DSBs) are the most detrimental form of DNA damage, and nonhomologous end-joining (NHEJ) mechanisms play dominant and priority roles in initiating DSB repair. A well-studied oncogene, the ubiquitin ligase Cullin 4A (CUL4A), is reported to be recruited to DSB sites in genomic DNA, but whether it regulates NHEJ mechanisms of DSB repair is unclear. Here, we discovered that the CUL4A-DTL ligase complex targeted the DNA-PKcs protein in the NHEJ repair pathway for nuclear degradation. Overexpression of either CUL4A or DTL reduced NHEJ repair efficiency and subsequently increased the accumulation of DSBs. Moreover, we demonstrated that overexpression of either CUL4A or DTL in normal cells led to genomic instability and malignant proliferation. Consistent with the in vitro findings, in human precancerous lesions, CUL4A expression gradually increased with increasing malignant tendency and was negatively correlated with DNA-PKcs and positively correlated with γ-H2AX expression. Collectively, this study provided strong evidence that the CUL4A-DTL axis increases genomic instability and enhances the subsequent malignant transformation of normal cells by inhibiting NHEJ repair. These results also suggested that CUL4A may be a prognostic marker of precancerous lesions and a potential therapeutic target in cancer.
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Leroy, Sabine, Isabelle Lebert, Carine Andant, Pierre Micheau, and Régine Talon. "Investigating Extracellular DNA Release in Staphylococcus xylosus Biofilm In Vitro." Microorganisms 9, no. 11 (October 21, 2021): 2192. http://dx.doi.org/10.3390/microorganisms9112192.
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Staphylococcus xylosus forms biofilm embedded in an extracellular polymeric matrix. As extracellular DNA (eDNA) resulting from cell lysis has been found in several staphylococcal biofilms, we investigated S. xylosus biofilm in vitro by a microscopic approach and identified the mechanisms involved in cell lysis by a transcriptomic approach. Confocal laser scanning microscopy (CLSM) analyses of the biofilms, together with DNA staining and DNase treatment, revealed that eDNA constituted an important component of the matrix. This eDNA resulted from cell lysis by two mechanisms, overexpression of phage-related genes and of cidABC encoding a holin protein that is an effector of murein hydrolase activity. This lysis might furnish nutrients for the remaining cells as highlighted by genes overexpressed in nucleotide salvage, in amino sugar catabolism and in inorganic ion transports. Several genes involved in DNA/RNA repair and genes encoding proteases and chaperones involved in protein turnover were up-regulated. Furthermore, S. xylosus perceived osmotic and oxidative stresses and responded by up-regulating genes involved in osmoprotectant synthesis and in detoxification. This study provides new insight into the physiology of S. xylosus in biofilm.
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Caudell, David L., and Abdul Gafoor A. Puthiyaveetil. "The Leukemic Fusion Gene NUP98-HOXD13 Suppresses Non-Homologous End Joining Factors and Impairs DNA Double Strand Break Repair." Blood 120, no. 21 (November 16, 2012): 521. http://dx.doi.org/10.1182/blood.v120.21.521.521.
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Abstract Abstract 521 Chromosomal translocations are hallmark features of hematologic malignancies that often require collaborating mutations for malignant transformation. These secondary mutations can occur spontaneously, or rather, be induced by the primary translocation. Mutations can occur as a result of DNA damage and misrepair with DNA double strand breaks being one of the most serious types of cell damage. Double strand breaks are classically repaired by the non-homologous end joining (NHEJ) mechanism and impaired NHEJ has been shown to promote mutagenesis. Transgenic mice expressing the myeloid leukemic fusion gene NUP98-HOXD13 (NHD13) develop Myelodysplastic syndrome and progress to acute leukemia after acquiring secondary mutations. Our studies have shown that B lymphocyte development and class switch recombination are impaired in stimulated B lymphocytes from NHD13 mice. Based on this, we used in vitro class switch recombination (CSR) to delineate the DNA break induction and repair mechanisms in NHD13 B lymphocytes. Naïve B lymphocytes were harvested from wild type (WT) and NHD13 spleens and cultured in the presence of E.coli Lipopolysaccharide (LPS) and IL-4 to induce CSR. The DNA break induction pattern was determined using phosphorylated H2AX labeling combined with confocal microscopy and flow cytometry. Our results showed that NHD13 B lymphocytes had a comparable break induction pattern, but significantly reduced DNA repair. Analysis of the cell cycle pattern of stimulated B cells at 24 hour intervals showed cell cycle arrest at the G2/M phase at 72 hours following stimulation—a hallmark feature of impaired DNA break repair. We analyzed the expression of classical NHEJ and alternative end joining factors including Ku70, Ku80, DNA Protein Kinase catalytic subunit (DNAPKcs), Xrcc4, DNA ligase 4, Ligase 1, and Ligase 3. Our results showed reduced expression of DNAPKcs, Ligase 4 and Xrcc4 in NHD13 B lymphocytes at 72 hours following stimulation, suggesting that cells failed to initiate NHEJ-mediated DNA repair. Our results suggest that a myeloid leukemic gene can impair the DNA repair mechanism and may indirectly promote mutations necessary for malignant transformation. Disclosures: No relevant conflicts of interest to declare.
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Vilpo, J. A., L. M. Vilpo, D. E. Szymkowski, A. O'Donovan, and R. D. Wood. "An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells." Molecular and Cellular Biology 15, no. 1 (January 1995): 290–97. http://dx.doi.org/10.1128/mcb.15.1.290.
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One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.
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Leong, Merrin Man Long, Arthur Kwok Leung Cheung, Wei Dai, Sai Wah Tsao, Chi Man Tsang, Christopher W. Dawson, Josephine Mun Yee Ko, and Maria Li Lung. "EBV infection is associated with histone bivalent switch modifications in squamous epithelial cells." Proceedings of the National Academy of Sciences 116, no. 28 (June 24, 2019): 14144–53. http://dx.doi.org/10.1073/pnas.1821752116.
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Epstein−Barr virus (EBV) induces histone modifications to regulate signaling pathways involved in EBV-driven tumorigenesis. To date, the regulatory mechanisms involved are poorly understood. In this study, we show that EBV infection of epithelial cells is associated with aberrant histone modification; specifically, aberrant histone bivalent switches by reducing the transcriptional activation histone mark (H3K4me3) and enhancing the suppressive mark (H3K27me3) at the promoter regions of a panel of DNA damage repair members in immortalized nasopharyngeal epithelial (NPE) cells. Sixteen DNA damage repair family members in base excision repair (BER), homologous recombination, nonhomologous end-joining, and mismatch repair (MMR) pathways showed aberrant histone bivalent switches. Among this panel of DNA repair members,MLH1, involved in MMR, was significantly down-regulated in EBV-infected NPE cells through aberrant histone bivalent switches in a promoter hypermethylation-independent manner. Functionally, expression ofMLH1correlated closely with cisplatin sensitivity both in vitro and in vivo. Moreover, seven BER members with aberrant histone bivalent switches in the EBV-positive NPE cell lines were significantly enriched in pathway analysis in a promoter hypermethylation-independent manner. This observation is further validated by their down-regulation in EBV-infected NPE cells. The in vitro comet and apurinic/apyrimidinic site assays further confirmed that EBV-infected NPE cells showed reduced DNA damage repair responsiveness. These findings suggest the importance of EBV-associated aberrant histone bivalent switch in host cells in subsequent suppression of DNA damage repair genes in a methylation-independent manner.
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Lan, Jing, Wenjin Li, Feng Zhang, Feng-Yan Sun, Tetsuya Nagayama, Cristine O'Horo, and Jun Chen. "Inducible Repair of Oxidative DNA Lesions in the Rat Brain after Transient Focal Ischemia and Reperfusion." Journal of Cerebral Blood Flow & Metabolism 23, no. 11 (November 2003): 1324–39. http://dx.doi.org/10.1097/01.wcb.0000091540.60196.f2.
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To determine the role of oxidative DNA damage and repair in brain injury after focal ischemia and reperfusion, the authors investigated DNA base damage and DNA base excision repair (BER) capacity, the predominant repair mechanism for oxidative DNA lesions, in the rat model of temporary middle cerebral artery occlusion. Contents of 8-hydroxyl-2′-deoxyguanosine (8-oxodG) and apurinic/apyrimidinic abasic site (AP site), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains 0.25 to 72 hours after 1 hour of middle cerebral artery occlusion. In parallel to the detection of DNA lesions, the capacity for 8-oxodG- or AP site-dependent DNA repair synthesis was measured in nuclear protein extracts using specific in vitro DNA repair assays. After postischemic reperfusion, the levels of 8-oxodG and AP sites were markedly increased in ischemic tissues. In frontal/parietal cortex, regions that survived ischemia, 8-oxodG and AP sites were efficiently repaired during reperfusion. However, in the caudate, a region that was destined to infarct, the DNA lesions were poorly repaired. In consistent with the patterns of endogenous lesion repair, a markedly induced and long-lasting (at least 72 hours) BER activity was detected in the cortex but not in the caudate after ischemia. The induced BER activity in ischemic cortex was attributed to the upregulation of gene expression and activation of selective BER enzymes, particularly DNA polymerase-β and OGG1. These results strongly suggest that inducible DNA BER constitutes an important endogenous mechanism that protects brain against ischemia-induced oxidative neuronal injury.
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Rawat, Vipin, Giulia Bortolussi, Silvia Gazzin, Claudio Tiribelli, and Andrés F. Muro. "Bilirubin-Induced Oxidative Stress Leads to DNA Damage in the Cerebellum of Hyperbilirubinemic Neonatal Mice and Activates DNA Double-Strand Break Repair Pathways in Human Cells." Oxidative Medicine and Cellular Longevity 2018 (November 26, 2018): 1–11. http://dx.doi.org/10.1155/2018/1801243.
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Unconjugated bilirubin is considered a potent antioxidant when present at moderate levels. However, at high concentrations, it produces severe neurological damage and death associated with kernicterus due to oxidative stress and other mechanisms. While it is widely recognized that oxidative stress by different toxic insults results in severe damage to cellular macromolecules, especially to DNA, no data are available either on DNA damage in the brain triggered by hyperbilirubinemia during the neonatal period or on the activation of DNA repair mechanisms. Here, using a mouse model of neonatal hyperbilirubinemia, we demonstrated that DNA damage occurs in vivo in the cerebellum, the brain region most affected by bilirubin toxicity. We studied the mechanisms associated with potential toxic action of bilirubin on DNA in in vitro models, which showed significant increases in DNA damage when neuronal and nonneuronal cells were treated with 140 nM of free bilirubin (Bf), as determined by γH2AX Western blot and immunofluorescence analyses. Cotreatment of cells with N-acetyl-cysteine, a potent oxidative-stress inhibitor, prevented DNA damage by bilirubin, supporting the concept that DNA damage was caused by bilirubin-induced oxidative stress. Bilirubin treatment also activated the main DNA repair pathways through homologous recombination (HR) and nonhomologous end joining (NHEJ), which may be adaptive responses to repair bilirubin-induced DNA damage. Since DNA damage may be another important factor contributing to neuronal death and bilirubin encephalopathy, these results contribute to the understanding of the mechanisms associated with bilirubin toxicity and may be of relevance in neonates affected with severe hyperbilirubinemia.
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Yi, Guo-zhong, Guanglong Huang, Manlan Guo, Xi’an Zhang, Hai Wang, Shengze Deng, Yaomin Li, et al. "Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2." Brain 142, no. 8 (July 25, 2019): 2352–66. http://dx.doi.org/10.1093/brain/awz202.
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Abstract The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.
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Sander, Peter, K. G. Papavinasasundaram, Thomas Dick, Evangelos Stavropoulos, Kerstin Ellrott, Burkhard Springer, M. Joseph Colston, and Erik C. Böttger. "Mycobacterium bovis BCG recADeletion Mutant Shows Increased Susceptibility to DNA-Damaging Agents but Wild-Type Survival in a Mouse Infection Model." Infection and Immunity 69, no. 6 (June 1, 2001): 3562–68. http://dx.doi.org/10.1128/iai.69.6.3562-3568.2001.
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ABSTRACT Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by theMycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovisBCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recAgenotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.
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Wang, Z., X. Wu, and E. C. Friedberg. "DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 2 (February 1993): 1051–58. http://dx.doi.org/10.1128/mcb.13.2.1051-1058.1993.
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Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents. We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts. Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway. Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures. However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon. Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair. Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract. These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.
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Wang, Z., X. Wu, and E. C. Friedberg. "DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 2 (February 1993): 1051–58. http://dx.doi.org/10.1128/mcb.13.2.1051.
Full textAbstract:
Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents. We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts. Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway. Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures. However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon. Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair. Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract. These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.
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Rogers, Cody M., Chun-Ying Lee, Samuel Parkins, Nicholas J. Buehler, Sabine Wenzel, Francisco Martínez-Márquez, Yuichiro Takagi, Sua Myong, and Matthew L. Bochman. "The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair." Journal of Biological Chemistry 295, no. 27 (May 5, 2020): 8945–57. http://dx.doi.org/10.1074/jbc.ra120.013626.
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DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)– and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro. We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.
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Hu, Minhao, Yiyun Lou, Shuyuan Liu, Yuchan Mao, Fang Le, Liya Wang, Lejun Li, et al. "Altered expression of DNA damage repair genes in the brain tissue of mice conceived by in vitro fertilization." Molecular Human Reproduction 26, no. 3 (January 31, 2020): 141–53. http://dx.doi.org/10.1093/molehr/gaaa010.
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Abstract Our previous study revealed a higher incidence of gene dynamic mutation in newborns conceived by IVF, highlighting that IVF may be disruptive to the DNA stability of IVF offspring. However, the underlying mechanisms remain unclear. The DNA damage repair system plays an essential role in gene dynamic mutation and neurodegenerative disease. To evaluate the long-term impact of IVF on DNA damage repair genes, we established an IVF mouse model and analyzed gene and protein expression levels of MSH2, MSH3, MSH6, MLH1, PMS2, OGG1, APEX1, XPA and RPA1 and also the amount of H2AX phosphorylation of serine 139 which is highly suggestive of DNA double-strand break (γH2AX expression level) in the brain tissue of IVF conceived mice and their DNA methylation status using quantitative real-time PCR, western blotting and pyrosequencing. Furthermore, we assessed the capacity of two specific non-physiological factors in IVF procedures during preimplantation development. The results demonstrated that the expression and methylation levels of some DNA damage repair genes in the brain tissue of IVF mice were significantly changed at 3 weeks, 10 weeks and 1.5 years of age, when compared with the in vivo control group. In support of mouse model findings, oxygen concentration of in vitro culture environment was shown to have the capacity to modulate gene expression and DNA methylation levels of some DNA damage repair genes. In summary, our study indicated that IVF could bring about long-term alterations of gene and protein expression and DNA methylation levels of some DNA damage repair genes in the brain tissue and these alterations might be resulted from the different oxygen concentration of culture environment, providing valuable perspectives to improve the safety and efficiency of IVF at early embryonic stage and also throughout different life stages.
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Hu, Jinchuan, Jun-Hyuk Choi, Shobhan Gaddameedhi, Michael G. Kemp, Joyce T. Reardon, and Aziz Sancar. "Nucleotide Excision Repair in Human Cells." Journal of Biological Chemistry 288, no. 29 (June 8, 2013): 20918–26. http://dx.doi.org/10.1074/jbc.m113.482257.
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Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.
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Ramilo, Cecilia, Liya Gu, Shuangli Guo, Xiping Zhang, Steve M. Patrick, John J. Turchi, and Guo-Min Li. "Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A." Molecular and Cellular Biology 22, no. 7 (April 1, 2002): 2037–46. http://dx.doi.org/10.1128/mcb.22.7.2037-2046.2002.
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ABSTRACT DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.
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Muller, Catherine, Garyfallia Christodoulopoulos, Bernard Salles, and Lawrence Panasci. "DNA-Dependent Protein Kinase Activity Correlates With Clinical and In Vitro Sensitivity of Chronic Lymphocytic Leukemia Lymphocytes to Nitrogen Mustards." Blood 92, no. 7 (October 1, 1998): 2213–19. http://dx.doi.org/10.1182/blood.v92.7.2213.
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Abstract The objective of this study is to investigate the role of DNA-dependent protein kinase (DNA-PK) in the chronic lymphocytic leukemia (CLL) lymphocyte response to nitrogen mustard therapy. DNA-PK is a nuclear serine/threonine kinase that functions in DNA double-strand break repair and in the joining process in recombination mechanisms. In a series of 34 patients with B-CLL, either untreated (n = 16) or resistant to chlorambucil (n = 18), the kinase activity of the complex, as determined by its capacity to phosphorylate a peptide substrate in vitro, is increased in the resistant samples as compared with the untreated ones (24.4 ± 2.6 arbitrary units [a.u.] [range, 12.7 to 55.8 a.u.] versus 8.1 ± 2.8 a.u. [range, 0.9 to 44.5 a.u.], respectively (P < .0001]), independent of other clinical and biological factors. Linear regression analysis shows an excellent correlation between the level of DNA-PK activity and the inherent in vitro sensitivity of CLL lymphocytes to chlorambucil (r = .875, P =.0001). The regulation of DNA-PK activity was associated with increased DNA-binding activity of its regulatory subunit, the Ku heterodimer, in resistant samples. These results suggest that this activity is a determinant in the cellular response to chlorambucil and participates in the development of nitrogen mustard–resistant disease. The increase in DNA-PK activity might contribute to the enhanced cross-link repair that we previously postulated to be a primary mechanism of resistance to nitrogen mustards in CLL.
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Muller, Catherine, Garyfallia Christodoulopoulos, Bernard Salles, and Lawrence Panasci. "DNA-Dependent Protein Kinase Activity Correlates With Clinical and In Vitro Sensitivity of Chronic Lymphocytic Leukemia Lymphocytes to Nitrogen Mustards." Blood 92, no. 7 (October 1, 1998): 2213–19. http://dx.doi.org/10.1182/blood.v92.7.2213.2213_2213_2219.
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The objective of this study is to investigate the role of DNA-dependent protein kinase (DNA-PK) in the chronic lymphocytic leukemia (CLL) lymphocyte response to nitrogen mustard therapy. DNA-PK is a nuclear serine/threonine kinase that functions in DNA double-strand break repair and in the joining process in recombination mechanisms. In a series of 34 patients with B-CLL, either untreated (n = 16) or resistant to chlorambucil (n = 18), the kinase activity of the complex, as determined by its capacity to phosphorylate a peptide substrate in vitro, is increased in the resistant samples as compared with the untreated ones (24.4 ± 2.6 arbitrary units [a.u.] [range, 12.7 to 55.8 a.u.] versus 8.1 ± 2.8 a.u. [range, 0.9 to 44.5 a.u.], respectively (P < .0001]), independent of other clinical and biological factors. Linear regression analysis shows an excellent correlation between the level of DNA-PK activity and the inherent in vitro sensitivity of CLL lymphocytes to chlorambucil (r = .875, P =.0001). The regulation of DNA-PK activity was associated with increased DNA-binding activity of its regulatory subunit, the Ku heterodimer, in resistant samples. These results suggest that this activity is a determinant in the cellular response to chlorambucil and participates in the development of nitrogen mustard–resistant disease. The increase in DNA-PK activity might contribute to the enhanced cross-link repair that we previously postulated to be a primary mechanism of resistance to nitrogen mustards in CLL.
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Karim, O. M., K. Pienta, N. Seki, and J. L. Mostwin. "Stretch-mediated visceral smooth muscle growth in vitro." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 262, no. 5 (May 1, 1992): R895—R900. http://dx.doi.org/10.1152/ajpregu.1992.262.5.r895.
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An in vitro model of smooth muscle stretch was developed to study mechanical stimulus as a possible mediator of visceral smooth muscle growth and differences in the growth response of smooth muscle from young and old animals. De novo DNA synthesis as measured by the aphidicolin-sensitive specific activity of DNA was used as an index of cell growth. Compared with old tissue, the rate of aphidicolin-sensitive DNA synthesis in smooth muscle from young animals was 3-5 and 1.5-2 times greater in bladder and taenia coli, respectively. Stretch of bladder muscle and taenia coli strips from young animals for 6 h increased the aphidicolin-sensitive specific activity of DNA 3-fold (P less than 0.01) and 1.5-fold (P less than 0.01), respectively. Tissue from old animals, however, under the same conditions increased the rate of aphidicolin-resistant DNA synthesis, possibly implying DNA repair. Autoradiography showed only labeled myocyte nuclei. These results indicate that homeostatic mechanisms modulating myocyte growth in visceral smooth muscle can respond to mechanical stimulus in the absence of other trophic factors.
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Soulas-Sprauel, Pauline, Gwenaël Le Guyader, Paola Rivera-Munoz, Vincent Abramowski, Christelle Olivier-Martin, Cécile Goujet-Zalc, Pierre Charneau, and Jean-Pierre de Villartay. "Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination." Journal of Experimental Medicine 204, no. 7 (July 2, 2007): 1717–27. http://dx.doi.org/10.1084/jem.20070255.
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V(D)J recombination and immunoglobulin class switch recombination (CSR) are two somatic rearrangement mechanisms that proceed through the introduction of double-strand breaks (DSBs) in DNA. Although the DNA repair factor XRCC4 is essential for the resolution of DNA DSB during V(D)J recombination, its role in CSR has not been established. To bypass the embryonic lethality of XRCC4 deletion in mice, we developed a conditional XRCC4 knockout (KO) using LoxP-flanked XRCC4 cDNA lentiviral transgenesis. B lymphocyte restricted deletion of XRCC4 in these mice lead to an average two-fold reduction in CSR in vivo and in vitro. Our results connect XRCC4 and the nonhomologous end joining DNA repair pathway to CSR while reflecting the possible use of an alternative pathway in the repair of CSR DSB in the absence of XRCC4. In addition, this new conditional KO approach should be useful in studying other lethal mutations in mice.
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Yu, Man, and Warren Masker. "T7 Single Strand DNA Binding Protein but Not T7 Helicase Is Required for DNA Double Strand Break Repair." Journal of Bacteriology 183, no. 6 (March 15, 2001): 1862–69. http://dx.doi.org/10.1128/jb.183.6.1862-1869.2001.
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ABSTRACT An in vitro system based on Escherichia coliinfected with bacteriophage T7 was used to test for involvement of host and phage recombination proteins in the repair of double strand breaks in the T7 genome. Double strand breaks were placed in a uniqueXhoI site located approximately 17% from the left end of the T7 genome. In one assay, repair of these breaks was followed by packaging DNA recovered from repair reactions and determining the yield of infective phage. In a second assay, the product of the reactions was visualized after electrophoresis to estimate the extent to which the double strand breaks had been closed. Earlier work demonstrated that in this system double strand break repair takes place via incorporation of a patch of DNA into a gap formed at the break site. In the present study, it was found that extracts prepared from uninfected E. coli were unable to repair broken T7 genomes in this in vitro system, thus implying that phage rather than host enzymes are the primary participants in the predominant repair mechanism. Extracts prepared from an E. coli recA mutant were as capable of double strand break repair as extracts from a wild-type host, arguing that the E. coli recombinase is not essential to the recombinational events required for double strand break repair. In T7 strand exchange during recombination is mediated by the combined action of the helicase encoded by gene 4 and the annealing function of the gene 2.5 single strand binding protein. Although a deficiency in the gene 2.5 protein blocked double strand break repair, a gene 4 deficiency had no effect. This argues that a strand transfer step is not required during recombinational repair of double strand breaks in T7 but that the ability of the gene 2.5 protein to facilitate annealing of complementary single strands of DNA is critical to repair of double strand breaks in T7.
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Kucuker, M., A. Ozerdem, D. Ceylan, A. Cabello-Arreola, M. C. Ho, B. Joseph, L. Webb, P. Croarkin, M. Frye, and M. Veldic. "The Role of Base Excision Repair in Major Depressive Disorder and Bipolar Disorder." European Psychiatry 65, S1 (June 2022): S401. http://dx.doi.org/10.1192/j.eurpsy.2022.1016.
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Introduction In vivo and in vitro studies suggest that inflammation and oxidative damage may contribute to the pathogenesis of major depressive disorder (MDD) and bipolar disorder (BD). Imbalance between DNA damage and repair is an emerging research area examining pathophysiological mechanisms of these major mood disorders. Objectives This systematic review sought to examine current evidence on the association between mood disorders and deficits in base excision repair (BER), the primary repair mechanism for repair of oxidation-induced DNA lesions. Methods We conducted a comprehensive literature search of Ovid MEDLINE® Epub Ahead of Print, Ovid MEDLINE® In-Process & Other Non-Indexed Citations, Ovid MEDLINE® Daily, EMBASE (1947), and PsycINFO for studies investigating the alterations in base excision repair in patients with MDD or BD. Results A total of 1,364 records were identified. 1,352 records remained after duplicates were removed. 24 records were selected for full-text screening and a remaining 12 articles were included in the qualitative synthesis. SNPs (Single Nucleotide Polymorphisms) of several BER genes have been shown to be associated with MDD and BD. However, it was difficult to draw conclusions from BER gene expression studies due to conflicting findings and the small number of studies. Conclusions Future studies comparing DNA repair during the manic or depressive episode to remission will give us a better insight regarding the role of DNA repair in mood disorders. These alterations might be utilized as diagnostic and prognostic biomarkers as well as measuring treatment response. Disclosure No significant relationships.
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Fanale, Daniele, Viviana Bazan, Stefano Caruso, Marta Castiglia, Giuseppe Bronte, Christian Rolfo, Giuseppe Cicero, and Antonio Russo. "Hypoxia and Human Genome Stability: Downregulation of BRCA2 Expression in Breast Cancer Cell Lines." BioMed Research International 2013 (2013): 1–8. http://dx.doi.org/10.1155/2013/746858.
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Previously, it has been reported that hypoxia causes increased mutagenesis and alteration in DNA repair mechanisms. In 2005, an interesting study showed that hypoxia-induced decreases in BRCA1 expression and the consequent suppression of homologous recombination may lead to genetic instability. However, nothing is yet known about the involvement of BRCA2 in hypoxic conditions in breast cancer. Initially, a cell proliferation assay allowed us to hypothesize that hypoxia could negatively regulate the breast cancer cell growth in short term in vitro studies. Subsequently, we analyzed gene expression in breast cancer cell lines exposed to hypoxic condition by microarray analysis. Interestingly, genes involved in DNA damage repair pathways such as mismatch repair, nucleotide excision repair, nonhomologous end-joining and homologous recombination repair were downregulated. In particular, we focused on the BRCA2 downregulation which was confirmed at mRNA and protein level. In addition, breast cancer cells were treated with dimethyloxalylglycine (DMOG), a cell-permeable inhibitor of both proline and asparaginyl hydroxylases able to induce HIF-1αstabilization in normoxia, providing results comparable to those previously described. These findings may provide new insights into the mechanisms underlying genetic instability mediated by hypoxia and BRCA involvement in sporadic breast cancers.
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Scherzad, Agmal, Till Meyer, Norbert Kleinsasser, and Stephan Hackenberg. "Molecular Mechanisms of Zinc Oxide Nanoparticle-Induced Genotoxicity." Materials 10, no. 12 (December 14, 2017): 1427. http://dx.doi.org/10.3390/ma10121427.
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Background: Zinc oxide nanoparticles (ZnO NPs) are among the most frequently applied nanomaterials in consumer products. Evidence exists regarding the cytotoxic effects of ZnO NPs in mammalian cells; however, knowledge about the potential genotoxicity of ZnO NPs is rare, and results presented in the current literature are inconsistent. Objectives: The aim of this review is to summarize the existing data regarding the DNA damage that ZnO NPs induce, and focus on the possible molecular mechanisms underlying genotoxic events. Methods: Electronic literature databases were systematically searched for studies that report on the genotoxicity of ZnO NPs. Results: Several methods and different endpoints demonstrate the genotoxic potential of ZnO NPs. Most publications describe in vitro assessments of the oxidative DNA damage triggered by dissoluted Zn2+ ions. Most genotoxicological investigations of ZnO NPs address acute exposure situations. Conclusion: Existing evidence indicates that ZnO NPs possibly have the potential to damage DNA. However, there is a lack of long-term exposure experiments that clarify the intracellular bioaccumulation of ZnO NPs and the possible mechanisms of DNA repair and cell survival.
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Zamansky, G. B., D. F. Minka, C. L. Deal, and K. Hendricks. "The in vitro photosensitivity of systemic lupus erythematosus skin fibroblasts." Journal of Immunology 134, no. 3 (March 1, 1985): 1571–76. http://dx.doi.org/10.4049/jimmunol.134.3.1571.
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Abstract To investigate the role of DNA damage in the pathogenesis of systemic lupus erythematosus (SLE), we studied the ability of skin fibroblasts derived from SLE patients to recover from ultraviolet (UV) light radiation of varying wavelengths. Four of five SLE cell strains were more sensitive to UV-C (254 nm), sun lamp, and UV-A (320 to 400 nm) light than were normal cells. SLE cellular recovery was most sensitive to broad spectrum, long wavelength light. This hypersensitivity did not appear to result from the UV light activation of a clastogenic factor. Experiments which examined the DNA repair capacity of irradiated cells indicated that SLE fibroblasts may be able to excise certain DNA lesions as well as normal cells. The mechanisms responsible for the hypersensitivity of SLE cells remain under investigation.
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Villablanca, Amparo C. "Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro." Journal of Applied Physiology 84, no. 6 (June 1, 1998): 2089–98. http://dx.doi.org/10.1152/jappl.1998.84.6.2089.
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Nicotine is a major component of cigarette smoke and has been postulated to play an important role in atherogenesis and malignancy. Endothelial cell growth may be regulated by nicotine, yet operative mechanisms at the endothelial level are poorly understood. We studied the effects of nicotine (10−14-10−4M) on endothelial DNA synthesis, DNA repair, proliferation, and cytotoxicity by using cultures of bovine pulmonary artery endothelial cells. Assays were performed on cells incubated with nicotine in the presence and absence of hydroxyurea (an inhibitor of scheduled DNA synthesis), serum, human platelet-poor plasma, and platelet-derived growth factor and endothelial cell growth factor (PDGF and PDECGF, respectively). Nicotine significantly stimulated endothelial cell DNA synthesis and proliferation at concentrations lower than those obtained in blood after smoking (<10−8M). The stimulatory effects of nicotine were enhanced by serum (0.5%) and PDECGF and were blocked by the nicotinic-receptor antagonist hexamethonium. The response to nicotine was bimodal because cytotoxicity was observed at higher concentrations (>10−6M). This study has implications for understanding cellular mechanisms of nicotine action. The results may be important in tumor angiogenesis, atherogenesis, and vascular dysfunction in smokers.
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Franchet, Camille, Jean-Sébastien Hoffmann, and Florence Dalenc. "Recent Advances in Enhancing the Therapeutic Index of PARP Inhibitors in Breast Cancer." Cancers 13, no. 16 (August 17, 2021): 4132. http://dx.doi.org/10.3390/cancers13164132.
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As poly-(ADP)-ribose polymerase (PARP) inhibition is synthetic lethal with the deficiency of DNA double-strand (DSB) break repair by homologous recombination (HR), PARP inhibitors (PARPi) are currently used to treat breast cancers with mutated BRCA1/2 HR factors. Unfortunately, the increasingly high rate of PARPi resistance in clinical practice has dented initial hopes. Multiple resistance mechanisms and acquired vulnerabilities revealed in vitro might explain this setback. We describe the mechanisms and vulnerabilities involved, including newly identified modes of regulation of DSB repair that are now being tested in large cohorts of patients and discuss how they could lead to novel treatment strategies to improve the therapeutic index of PARPi.
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Fabijańska, Małgorzata, Maria M. Kasprzak, and Justyn Ochocki. "Ruthenium(II) and Platinum(II) Complexes with Biologically Active Aminoflavone Ligands Exhibit In Vitro Anticancer Activity." International Journal of Molecular Sciences 22, no. 14 (July 15, 2021): 7568. http://dx.doi.org/10.3390/ijms22147568.
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Continuing our studies on the mechanisms underlying the cytotoxicity of potential drugs, we have described several aspects of the in vitro anticancer activity of ruthenium(II) and platinum(II) complexes with bioactive, synthetic aminoflavone ligands. We examined the mechanism of proapoptotic activity of cis-dichlorobis(3-imino-2-methoxyflavanone)ruthenium(II), cis-dichlorobis(3-imino-2-ethoxyflavanone)ruthenium(II), and trans-dichlorobis(3-aminoflavone)platinum(II). Cisplatin was used as a reference compound. The cytotoxicity was investigated by MTT assay. The mechanism of proapoptotic activity of the tested compounds was investigated by evaluation of caspase-8 activity, cytometric analysis of annexin-V positive cells, and mitochondrial potential loss measurement. The results showed that ruthenium compounds break partially or completely the cisplatin resistance by activating the caspase 8-dependent apoptosis pathway and loss of mitochondrial membrane potential. Platinum compounds also have a cytostatic effect, but their action requires more exposure time. Potential mechanisms underlying drug resistance in the two pairs of cancer cell lines were investigated: total glutathione content, P-glycoprotein activity, and differences in the activity of DNA repair induced by nucleotide excision. Results showed that cisplatin-resistant cells have elevated glutathione levels relative to sensitive cells. Moreover, they indicated the mechanisms enabling cells to avoid apoptosis caused by DNA damage. Pg-P activity has no effect on the development of cisplatin resistance in the cell lines described.
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San Juan, Lucía, Isabel de Pedro, Azahara Rodríguez-Luna, María Villalba, Antonio Guerrero, Salvador González, and Alberto Gandarillas. "Cryptomphalus aspersa Eggs Extract Potentiates Human Epidermal Stem Cell Regeneration and Amplification." Cosmetics 9, no. 1 (December 23, 2021): 2. http://dx.doi.org/10.3390/cosmetics9010002.
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Modern life and extended life expectancy have prompted the search for natural compounds alleviating skin aging. Evidence supports the beneficial effects on skin integrity and health from the topical administration of preparations of the mollusc Cryptomphalus aspersa eggs extract (IFC-CAF®) and suggests these effects are partly derived from an impact on skin renewal and repair mechanisms. The objective was to dissect in vitro the specific impact of IFC-CAF® on different parameters related to the regenerative potential, differentiation phenotype and exhaustion of skin stem cells. A prominent impact of IFC-CAF® was the induction of stratification and differentiated phenotypes from skin stem cells. IFC-CAF® slowed down the cell cycle at the keratinocyte DNA repair phase and, decelerated proliferation. However, it preserved the proliferative potential of the stem cells. IFC-CAF® reduced the DNA damage marker, γH2AX, and induced the expression of the transcription factor p53. These features correlated with significant protection in telomere shortening upon replicative exhaustion. Thus, IFC-CAF® helps maintain orderly cell cycling and differentiation, thus potentiating DNA repair and integrity. Our observations support the regenerative and repair capacity of IFC-CAF® on skin, through the improved mobilization and ordered differentiation of keratinocyte precursors and the enhancement of genome surveillance and repair mechanisms that counteract aging.
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Kitagawa, Takao, Hisashi Hoshida, and Rinji Akada. "Genome-Wide Analysis of Cellular Response to Bacterial Genotoxin CdtB in Yeast." Infection and Immunity 75, no. 3 (January 12, 2007): 1393–402. http://dx.doi.org/10.1128/iai.01321-06.
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ABSTRACT The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by several bacterial pathogens, and the subunit CdtB has the ability to create DNA lesions, primarily DNA single-strand breaks (SSBs) in vitro, and cause cell cycle arrest, cellular distension, and cell death in both mammalian and yeast cells. To elucidate the components of the mechanisms underlying the response to CdtB-induced DNA lesions, a CdtB expression plasmid was transformed into a series of diploid yeast strains harboring deletions in 4,708 nonessential genes. A total of 4,706 of these clones were successfully transformed, which we have now designated as a systematic transformation array (STA), and were subsequently screened. We identified 61 sensitive strains from the STA whose deleted genes can be categorized into a number of groups, including DNA metabolism, chromosome segregation, vesicular traffic, RNA catabolism, protein translation, morphogenesis, and nuclear transport, as well as one unknown open reading frame. However, only 28 of these strains were found to be sensitive to HO endonuclease, which is known to create a DNA double-strand break (DSB), suggesting that CdtB-induced DNA lesion is not similar to the direct DSB. Amazingly, CdtB expression elicits severe growth defects in haploid yeast cells, but only marginal defects in diploid yeast cells. The presence and absence of genes known to be involved in DNA repair in these genome-wide data reveal that CdtB-induced DNA damage is specifically repaired well in the diploid by homologous recombination but not by other repair mechanisms. Our present results provide insights into how CdtB pathogenesis is linked to eukaryotic cellular functions.
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Przewloka, Marcin R., Paige E. Pardington, Steven M. Yannone, David J. Chen, and Robert B. Cary. "In Vitro and In Vivo Interactions of DNA Ligase IV with a Subunit of the Condensin Complex." Molecular Biology of the Cell 14, no. 2 (February 2003): 685–97. http://dx.doi.org/10.1091/mbc.e01-11-0117.
Full textAbstract:
Several findings have revealed a likely role for DNA ligase IV, and interacting protein XRCC4, in the final steps of mammalian DNA double-strand break repair. Recent evidence suggests that the human DNA ligase IV protein plays a critical role in the maintenance of genomic stability. To identify protein–protein interactions that may shed further light on the molecular mechanisms of DSB repair and the biological roles of human DNA ligase IV, we have used the yeast two-hybrid system in conjunction with traditional biochemical methods. These efforts have resulted in the identification of a physical association between the DNA ligase IV polypeptide and the human condensin subunit known as hCAP-E. The hCAP-E polypeptide, a member of the Structural Maintenance of Chromosomes (SMC) super-family of proteins, coimmunoprecipitates from cell extracts with DNA ligase IV. Immunofluorescence studies reveal colocalization of DNA ligase IV and hCAP-E in the interphase nucleus, whereas mitotic cells display colocalization of both polypeptides on mitotic chromosomes. Strikingly, the XRCC4 protein is excluded from the area of mitotic chromosomes, suggesting the formation of specialized DNA ligase IV complexes subject to cell cycle regulation. We discuss our findings in light of known and hypothesized roles for ligase IV and the condensin complex.
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Cockram, Charlotte A., Milana Filatenkova, Vincent Danos, Meriem El Karoui, and David R. F. Leach. "Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo." Proceedings of the National Academy of Sciences 112, no. 34 (August 10, 2015): E4735—E4742. http://dx.doi.org/10.1073/pnas.1424269112.
Full textAbstract:
Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome ofEscherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at thelacZlocus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.
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Sabirzhanov, Boris, Oleg Makarevich, James P. Barrett, Isabel L. Jackson, Ethan P. Glaser, Alan I. Faden, and Bogdan A. Stoica. "Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways." International Journal of Molecular Sciences 21, no. 15 (July 23, 2020): 5239. http://dx.doi.org/10.3390/ijms21155239.
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Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy.
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Chen, Shih-Hsun, and Xiaochun Yu. "Targeting dePARylation selectively suppresses DNA repair–defective and PARP inhibitor–resistant malignancies." Science Advances 5, no. 4 (April 2019): eaav4340. http://dx.doi.org/10.1126/sciadv.aav4340.
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While poly(ADP-ribosyl)ation (PARylation) plays an important role in DNA repair, the role of dePARylation in DNA repair remains elusive. Here, we report that a novel small molecule identified from the NCI database, COH34, specifically inhibits poly(ADP-ribose) glycohydrolase (PARG), the major dePARylation enzyme, with nanomolar potency in vitro and in vivo. COH34 binds to the catalytic domain of PARG, thereby prolonging PARylation at DNA lesions and trapping DNA repair factors. This compound induces lethality in cancer cells with DNA repair defects and exhibits antitumor activity in xenograft mouse cancer models. Moreover, COH34 can sensitize tumor cells with DNA repair defects to other DNA-damaging agents, such as topoisomerase I inhibitors and DNA-alkylating agents, which are widely used in cancer chemotherapy. Notably, COH34 also efficiently kills PARP inhibitor–resistant cancer cells. Together, our study reveals the molecular mechanism of PARG in DNA repair and provides an effective strategy for future cancer therapies.
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Richard, Guy-Franck. "The Startling Role of Mismatch Repair in Trinucleotide Repeat Expansions." Cells 10, no. 5 (April 26, 2021): 1019. http://dx.doi.org/10.3390/cells10051019.
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Trinucleotide repeats are a peculiar class of microsatellites whose expansions are responsible for approximately 30 human neurological or developmental disorders. The molecular mechanisms responsible for these expansions in humans are not totally understood, but experiments in model systems such as yeast, transgenic mice, and human cells have brought evidence that the mismatch repair machinery is involved in generating these expansions. The present review summarizes, in the first part, the role of mismatch repair in detecting and fixing the DNA strand slippage occurring during microsatellite replication. In the second part, key molecular differences between normal microsatellites and those that show a bias toward expansions are extensively presented. The effect of mismatch repair mutants on microsatellite expansions is detailed in model systems, and in vitro experiments on mismatched DNA substrates are described. Finally, a model presenting the possible roles of the mismatch repair machinery in microsatellite expansions is proposed.