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1.
ATM and Artemis promote homologous recombination of radiation‐induced DNA double‐strand breaks in G2 下载免费PDF全文
Olivia Barton Atsushi Shibata Sandro Conrad Aaron A Goodarzi Andrea Krempler Penny A Jeggo Markus Löbrich 《The EMBO journal》2009,28(21):3413-3427
Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ~15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ. 相似文献
2.
Ataxia telangiectasia mutated activation by transcription‐ and topoisomerase I‐induced DNA double‐strand breaks 下载免费PDF全文
Christophe E Redon Josée Guirouilh‐Barbat Susan Smith Stéphanie Solier Céline Douarre Chiara Conti Asako J Nakamura Benu B Das Estelle Nicolas Kurt W Kohn William M Bonner Yves Pommier 《EMBO reports》2009,10(8):887-893
3.
Maria Moreno‐Villanueva Alexander Bürkle Inge Petersen Vilhelm A. Bohr Kaare Christensen Tinna Stevnsner 《Aging cell》2013,12(1):58-66
Exogenous and endogenous damage to DNA is constantly challenging the stability of our genome. This DNA damage increase the frequency of errors in DNA replication, thus causing point mutations or chromosomal rearrangements and has been implicated in aging, cancer, and neurodegenerative diseases. Therefore, efficient DNA repair is vital for the maintenance of genome stability. The general notion has been that DNA repair capacity decreases with age although there are conflicting results. Here, we focused on potential age‐associated changes in DNA damage response and the capacities of repairing DNA single‐strand breaks (SSBs) and double‐strand breaks (DSBs) in human peripheral blood mononuclear cells (PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ‐H2AX response, and DSB repair capacity in a study population consisting of 216 individuals from a population‐based sample of twins aged 40–77 years. Age in this range did not seem to have any effect on the SSB parameters. However, γ‐H2AX response and DSB repair capacity decreased with increasing age, although the associations did not reach statistical significance after adjustment for batch effect across multiple experiments. No gender differences were observed for any of the parameters analyzed. Our findings suggest that in PBMCs, the repair of SSBs is maintained until old age, whereas the response to and the repair of DSBs decrease. 相似文献
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DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. 相似文献
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用简化的Kohn氏碱洗脱法,观察了光敏剂血卟啉衍生物(HPD)对小鼠S-180肿瘤细胞DNA单链断裂及其重接修复的影响。激光HPD能导致S-180细胞DNA单链断裂明显增加,而且这种断裂随着保温时间的延长,继续增多。在本实验条件下没有观察到HPD对X线的增敏作用,HPD不能增加X线所致的DNA单链断裂,也不能影响其重接。单链断裂重接动力学的实验进一步证明了这个论点。 相似文献
8.
本文将反向交变电场和六角形电极电场这两种脉冲电场凝胶电泳技术应用于X线照射小鼠乳癌细胞SR-1所致DNA双链断裂的检测,在本实验条件下,用这种电泳都能检测到低至1.5Gy照射所产生的DNA双链断裂,并且用六角形电极电场电泳获得了DNA双链断裂程度与照射剂量之间的良好线性关系,此外,还用此方法观察了不同浓度自由基清除剂DMSO对X线照射SR-1细胞所致DNA双链断裂的保护作用,结果进一步证实本方法的可靠性。 相似文献
9.
Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites,
base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand
breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more
biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects
of 137Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were
investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline
one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation
between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference
with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was
observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation,
and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired
24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
This investigation concerns with the effect of low intensity microwave (2.45 and 16.5 GHz, SAR 1.0 and 2.01 W/kg, respectively) radiation on developing rat brain. Wistar rats (35 days old, male, six rats in each group) were selected for this study. These animals were exposed for 35 days at the above mentioned frequencies separately in two different exposure systems. After the exposure period, the rats were sacrificed and the whole brain tissue was dissected and used for study of single strand DNA breaks by micro gel electrophoresis (comet assay). Single strand DNA breaks were measured as tail length of comet. Fifty cells from each slide and two slides per animal were observed. One-way ANOVA method was adopted for statistical analysis. This study shows that the chronic exposure to these radiations cause statistically significant (p<0.001) increase in DNA single strand breaks in brain cells of rat. 相似文献
11.
We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks. 相似文献
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Induction of DNA strand breaks by intermittent exposure to extremely-low-frequency electromagnetic fields in human diploid fibroblasts 总被引:11,自引:0,他引:11
Results of epidemiological research show low association of electromagnetic field (EMF) with increased risk of cancerous diseases and missing dose-effect relations. An important component in assessing potential cancer risk is knowledge concerning any genotoxic effects of extremely-low-frequency-EMF (ELF-EMF).Human diploid fibroblasts were exposed to continuous or intermittent ELF-EMF (50Hz, sinusoidal, 24h, 1000microT). For evaluation of genotoxic effects in form of DNA single- (SSB) and double-strand breaks (DSB), the alkaline and the neutral comet assay were used.In contrast to continuous ELF-EMF exposure, the application of intermittent fields reproducibly resulted in a significant increase of DNA strand break levels, mainly DSBs, as compared to non-exposed controls. The conditions of intermittence showed an impact on the induction of DNA strand breaks, producing the highest levels at 5min field-on/10min field-off. We also found individual differences in response to ELF-EMF as well as an evident exposure-response relationship between magnetic flux density and DNA migration in the comet assay.Our data strongly indicate a genotoxic potential of intermittent EMF. This points to the need of further studies in vivo and consideration about environmental threshold values for ELF exposure. 相似文献
14.
《Electromagnetic biology and medicine》2013,32(1):23-29
The effect of a temporally incoherent magnetic field noise on microwave-induced DNA single and double strand breaks in rat brain cells was investigated. Four treatment groups of rats were studied: microwave-exposure (continuous-wave 2450-MHz microwaves, power density 1 mW/cm2, average whole-body specific absorption rate of 0.6 W/kg), noise-exposure (45 mG), microwave + noise-exposure, and sham-exposure. Animals were exposed to these conditions for 2h. DNA single- and double-strand breaks in brain cells of these animals were assayed 4h later using a microgel electrophoresis assay. Results show that brain cells of microwave-exposed rats had significantly higher levels of DNA single- and double-strand breaks when compared with sham-exposed animals. Exposure to noise alone did not significantly affect the levels (i.e., they were similar to those of the sham-exposed rats). However, simultaneous noise exposure blocked microwave-induced increases in DNA strand breaks. These data indicate that simultaneous exposure to a temporally incoherent magnetic field could block microwave-induced DNA damage in brain cells of the rat. 相似文献
15.
Topoisomerase I‐mediated cleavage at unrepaired ribonucleotides generates DNA double‐strand breaks 下载免费PDF全文
Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2′,3′‐cyclic phosphates at ribonucleotide sites. Here, we provide genetic and biochemical evidence that DNA double‐strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. We show that RNase H2‐deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1‐induced damage at ribonuclelotide sites. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1‐induced DNA nicks at ribonucleotide sites. Analysis of Top1‐linked DNA from pull‐down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2‐deficient yeast cells, supporting this model. Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2. 相似文献
16.
Maintaining genome integrity in the germline is essential for survival and propagation of a species. In both mouse and human, germ cells originate during fetal development and are hypersensitive to both endogenous and exogenous DNA damaging agents. Currently, mechanistic understanding of how primordial germ cells respond to DNA damage is limited in part by the tools available to study these cells. We developed a mouse transgenic reporter strain expressing a 53BP1‐mCherry fusion protein under the control of the Oct4ΔPE embryonic germ cell‐specific promoter. This reporter binds sites of DNA double strand breaks (DSBs) on chromatin, forming foci. Using ionizing radiation as a DNA DSB‐inducing agent, we show that the transgenic reporter expresses specifically in the embryonic germ cells of both sexes and forms DNA damage induced foci in both a dose‐ and time‐dependent manner. The dynamic time‐sensitive and dose‐sensitive DNA damage detection ability of this transgenic reporter, in combination with its specific expression in embryonic germ cells, makes it a versatile and valuable tool for increasing our understanding of DNA damage responses in these unique cells. 相似文献
17.
SHLD2/FAM35A co‐operates with REV7 to coordinate DNA double‐strand break repair pathway choice 下载免费PDF全文
Vincent M Luo Abba Malina Théo Morin Yan Coulombe Billel Djerir Zhigang Li Arash Samiei Estelle Simo‐Cheyou Martin Karam Halil Bagci Dolev Rahat Damien Grapton Elise G Lavoie Christian Dove Husam Khaled Hellen Kuasne Koren K Mann Kathleen Oros Klein Celia M Greenwood Yuval Tabach Morag Park Jean‐Francois Côté Jean‐Yves Masson Alexandre Maréchal Alexandre Orthwein 《The EMBO journal》2018,37(18)
DNA double‐strand breaks (DSBs) can be repaired by two major pathways: non‐homologous end‐joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)‐based approach, we identify 11 high‐confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ‐mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1‐, RIF1‐, and REV7‐dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision‐making process during DSB repair. 相似文献
18.
The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the γ-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation. 相似文献
19.
Epicatechin (EC), a polyphenolic antioxidant compound found in tea, apples and chocolate offered protection to DNA against ionizing radiation induced damages. Under in vitro conditions of radiation exposure, plasmid pBR322 DNA was protected by EC in a concentration dependent manner. The dose modifying factor for 0.2 mM EC for 50% protection of the plasmid DNA was found to be 6.0. EC when administered to mice 1 h prior to exposure to 4 Gy gamma-radiation protected cellular DNA against radiation-induced strand breaks in peripheral blood leukocytes, as revealed in alkaline comet assay studies. Thus, EC was found to protect DNA from gamma-radiation indiced strand breaks under in vitro as well as in vivo conditions of radiation exposure. 相似文献
20.
Ursula M. Paredes Robert
Prys‐Jones Mark Adams Jim Groombridge Samit Kundu Paul‐Michael Agapow Richard L. Abel 《Journal of Zoological Systematics and Evolutionary Research》2012,50(3):247-250
Most zoological systematics studies are currently based on morphological features, molecular traits or a combination of both to reconstruct animals’ phylogenetic history. Increasingly, morphological studies of museum specimens are using X‐ray computed tomography to visualize internal morphology, because of its ‘non‐destructive’ nature. However, it is not known whether CT can fragment the size of DNA extracted from museum specimens, as has been demonstrated to occur in living cells. This question is of paramount importance for collections based research because X‐rays may reduce the amount of data obtainable from specimens. In our study, we tested whether exposure of museum bird skins to typical CT X‐ray energies (for visualization of the skeleton) increased DNA strand fragmentation, a key factor for the success of downstream molecular applications. For the present study, we extracted DNA from shavings of 24 prepared and dried bird skins (100+ years) footpads before and after CT scanning. The pre‐ and post‐CT fragmentation profiles were assessed using a capillary electrophoresis high‐precision instrument (Agilent Bioanalyzer). Comparison of the most common strand length in each DNA sample (relative mass) revealed no significant difference unexposed and exposed tissue (paired t‐test p = 0.463). In conclusion, we found no further quantifiable degradation of DNA strand length under standard X‐ray exposure obtained from our bird skins sample. Differences in museum preservation techniques probably had a greater effect on variation of pre‐CT DNA fragmentation. 相似文献