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1.
用脉冲电场凝胶电泳和双标记基因质粒DNA转染技术研究辐射敏感的毛细血管扩张性共济失调症患者皮肤成纤维细胞(AT5BIVA)和正常辐射抗性的人宫颈癌细胞(HeLaS3)DNA双链断裂重接修复率及其忠实性。结果表明γ射线照射诱发DNA双链断裂的产额和重接修复率,在两株细胞间无差别.而AT细胞对导入的限制性内切酶EcoRV产生双链断裂质粒DNA的重接修复忠实性显著低于HelaS3te胞,表明AT细胞易发生DNA错误修复,这很可能就是AT细胞高度辐射敏感性的主要原因。  相似文献   

2.
Ultraviolet light (UV) can provoke genome instability, partly through its ability to induce homologous recombination (HR). However, the mechanism(s) of UV-induced recombination is poorly understood. Although double-strand breaks (DSBs) have been invoked, there is little evidence for their generation by UV. Alternatively, single-strand DNA lesions that stall replication forks could provoke recombination. Recent findings suggest efficient initiation of UV-induced recombination in G1 through processing of closely spaced single-strand lesions to DSBs. However, other scenarios are possible, since the recombination initiated in G1 can be completed in the following stages of the cell cycle. We developed a system that could address UV-induced recombination events that start and finish in G2 by manipulating the activity of the sister chromatid cohesion complex. Here we show that sister-chromatid cohesion suppresses UV-induced recombination events that are initiated and resolved in G2. By comparing recombination frequencies and survival between UV and ionizing radiation, we conclude that a substantial portion of UV-induced recombination occurs through DSBs. This notion is supported by a direct physical observation of UV-induced DSBs that are dependent on nucleotide excision repair. However, a significant role of nonDSB intermediates in UV-induced recombination cannot be excluded.  相似文献   

3.
Ultraviolet light (UV) can provoke genome instability, partly through its ability to induce homologous recombination (HR). However, the mechanism(s) of UV-induced recombination is poorly understood. Although double-strand breaks (DSBs) have been invoked, there is little evidence for their generation by UV. Alternatively, single-strand DNA lesions that stall replication forks could provoke recombination. Recent findings suggest efficient initiation of UV-induced recombination in G1 through processing of closely spaced single-strand lesions to DSBs. However, other scenarios are possible, since the recombination initiated in G1 can be completed in the following stages of the cell cycle. We developed a system that could address UV-induced recombination events that start and finish in G2 by manipulating the activity of the sister chromatid cohesion complex. Here we show that sister-chromatid cohesion suppresses UV-induced recombination events that are initiated and resolved in G2. By comparing recombination frequencies and survival between UV and ionizing radiation, we conclude that a substantial portion of UV-induced recombination occurs through DSBs. This notion is supported by a direct physical observation of UV-induced DSBs that are dependent on nucleotide excision repair. However, a significant role of nonDSB intermediates in UV-induced recombination cannot be excluded.  相似文献   

4.
Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.  相似文献   

5.
    
This work describes a neutral and alkaline elution method for measuring DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-DNA crosslinks in rat testicular germ cells after treatments in vivo or in vitro with both chemical mutagens and gamma-irradiation. The methods depend upon the isolation of testicular germ cells by collagenase and trypsin digestion, followed by filtration and centrifugation.137Cs irradiation induced both DNA SSBs and DSBs in germ cells held on ice in vitro. Irradiation of the whole animal indicated that both types of DNA breaks are induced in vivo and can be repaired. A number of germ cell mutagens induced either DNA SSBs, DSBs, or cross-links after in vivo and in vitro dosing. These chemicals included methyl methane sulfonate, ethyl methane sulfonate, ethyl nitrosurea, dibromochlorpropane, ethylene dibromide, triethylene melamine, and mitomycin C. These results suggest that the blood-testes barrier is relatively ineffective for these mutagens, which may explain in part their in vivo mutagenic potency.This assay should be a useful screen for detecting chemical attack upon male germ-cell DNA and thus, it should help in the assessment of the mutagenic risk of chemicals. In addition, this approach can be used to study the processes of SSB, DSB, and crosslink repair in DNA of male germ cells, either from all stages or specific stages of development.Abbreviations DBCP dibromochlorpropane - DSB(s) DNA double-strand break(s) - EDB ethylene dibromide - EMS ethyl methane sulfonate - ENU ethyl nitrosurea - MC mitomycin C - MMS methyl methane sulfonate - SDS sodium dodecyl sulfate - SSB (s) DNA single-strand break(s) - TEM triethylene melamine - UDS unscheduled DNA synthesis  相似文献   

6.
Drosophila melanogaster has been widely used as a model organism to study various aspects of development. Apart from the whole Drosophila embryo, there are a number of cultured cell lines derived from Drosophila embryo that have also been used for elucidating various aspects of development. Drosophila Schneider line 2 cells were derived from the late stages of the embryo (Schneider, 1972). We found that the Schneider cells undergo myogenic differentiation upon treatment with neocarzinostatin (NCS), DNA double-strand break (DSB)-inducing drug, as indicated by elongated morphology, myosin heavy chain protein expression, multinucleation and exit from the cell cycle. No induction of differentiation was observed when cell proliferation was inhibited with drugs that do not cause DNA DSBs. Pre-treatment of Schneider cells with inhibitors of PKC, PP 1/2A, p38 MAPK, JNK and proteasomes resulted in the inhibition of morphological differentiation induced by NCS. These results indicate that DNA DSBs can turn on the myogenic program in Drosophila Schneider cells and the process is dependent on PK C-, PP 1/2A-, p38 MAPK-, and JNK- mediated signaling and proteasomal activity. The molting hormone, 20-hydroxyecdysone (20-HE), also showed an anti-myogenic effect on the process. This is the first report of insect cells undergoing differentiation by DNA DSB-inducing drugs as far as we know, and it provides a very useful and convenient in vitro system to study various aspects of Drosophila myogenesis.  相似文献   

7.
DNA链断裂检测技术的进展   总被引:6,自引:0,他引:6       下载免费PDF全文
DNA链损伤特别是DNA双链段裂(dsb)的检测方法是研究DNA辐射损伤的一个关键因素.已发展的检测DNA dsb的方法很多,但各种检测法均有其一定的优越性和适用范围,近年来应用较多并日益受到重视的新方法有原位杂交法,彗星试验(单细胞电泳法)以及高效毛细管电泳法等等.  相似文献   

8.
    
Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associa...  相似文献   

9.
10.
    
DNA ends pose specific problems in the control of genetic information quality. Ends of broken DNA need to be rejoined to avoid genome rearrangements, whereas natural DNA ends of linear chromosomes, telomeres, need to be stable and hidden from the DNA damage response. Efficient DNA end metabolism, either at induced DNA breaks or telomeres, does not result from the machine-like precision of molecular reactions, but rather from messier, more stochastic processes. The necessary molecular interactions are dynamically unstable, with constructive and destructive processes occurring in competition. In the end, quality control comes from the constant building up and tearing down of inappropriate, but also appropriate reaction steps in combination with factors that only slightly shift the equilibrium to eventually favour appropriate events. Thus, paradoxically, enzymes antagonizing DNA end metabolism help to ensure that genome maintenance becomes a robust process.  相似文献   

11.
The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced ‘closed’ conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5′ strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.  相似文献   

12.
    
DNA rearrangements, including inversions, translocations, and large insertions/deletions (indels), are crucial for crop evolution, domestication, and improvement. The rearrangements are frequently induced by ion beams via the mis-repair of DNA double-strand breaks (DSBs). Unfortunately, how ion beam-induced DSBs are repaired has not been comprehensively analyzed and the mechanisms underlying DNA rearrangements remain unclear. In this study, clonal sectors originating from single mutated cells in carbon ion-irradiated plants were used for whole-genome sequencing analyses after Arabidopsis seeds and seedlings were irradiated. Comparative analyses of the induced mutations (e.g., size and frequency of indels and microhomology at the junctions of the rearrangements) in the irradiated materials suggested that the broken/rejoined DSB ends were more extensively processed in seedlings than in seeds. A mutation to canonical non-homologous end-joining (c-NHEJ), which is a DSB repair pathway with minimal processing of DSB ends, increased the sensitivity to ion beams more in the seeds than in the seedlings, which was consistent with the junction analysis results, indicative of the minor contribution of c-NHEJ to the carbon ion-induced DSB repair in seedlings. Considering the characteristics of the large templated insertions in irradiated seedlings, ion-beam-induced DSBs in seedlings are likely repaired primarily by a polymerase theta-mediated pathway. Polymerase theta-deficient seedlings were more sensitive to ion beams than the c-NHEJ-deficient seedlings, consistent with this hypothesis. This study revealed the key characteristics of ion beam-induced DSBs and the associated repair mechanisms related to the physiological status of the irradiated materials, with implications for elucidating the occurrence and induction of rearrangements.  相似文献   

13.
SMU1是一个与细胞基因组复制和RNA剪切过程相关的新基因。该研究为进一步调查SMU1对细胞增殖及DNA双链断裂(DNAdouble—strand breaks,DNADSBs)损伤应答的影响,设计合成针对SMU1基因的小分子siRNA,并与对照siRNA(scramblel分别转染HEK293T或U2OS细胞。通过免疫印迹(Westernblot)检测证实,siSMU1转染细胞中SMU1的表达显著下降,采用台盼蓝染色细胞计数检测显示,SMU1表达下调显著降低细胞增殖能力。免疫荧光和免疫印迹法检测结果表明,SMU1表达下调显著增加细胞内源性DSBs损伤(7H2AXfoci和蛋白水平均升高);而进一步用X-ray处理细胞造成外源性DSBs损伤后,SMUI沉默细胞显示出延长的DSBs损伤修复动力学(减缓的γH2AXfoci和蛋白水平消退)。以上结果提示,SMU1在细胞DSBs损伤修复反应中扮演重要角色,积极参与细胞基因纽完整性的维持。  相似文献   

14.
    
The Artemis nuclease recognizes and endonucleolytically cleaves at single-stranded to double-stranded DNA (ss/dsDNA) boundaries. It is also a key enzyme in the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. Previously, a truncated form, Artemis-413, was developed that is constitutively active both in vitro and in vivo. Here, we use this constitutively active form of Artemis to detect DNA structures with ss/dsDNA boundaries that arise under topological stress. Topoisomerases prevent abnormal levels of torsional stress through modulation of positive and negative supercoiling. We show that overexpression of Artemis-413 in yeast cells carrying genetic mutations that ablate topoisomerase activity have an increased frequency of DNA double-strand breaks (DSBs). Based on the biochemical activity of Artemis, this suggests an increase in ss/dsDNA-containing structures upon increased torsional stress, with DSBs arising due to Artemis cutting at these ss/dsDNA structures. Camptothecin targets topoisomerase IB (Top1), and cells treated with camptothecin show increased DSBs. We find that expression of Artemis-413 in camptothecin-treated cells leads to a reduction in DSBs, the opposite of what we find with topoisomerase genetic mutations. This contrast between outcomes not only confirms that topoisomerase mutation and topoisomerase poisoning have distinct effects on cells, but also demonstrates the usefulness of Artemis-413 to study changes in DNA structure.  相似文献   

15.
Lupu A  Nevo E  Zamorzaeva I  Korol A 《Genetica》2006,127(1-3):121-132
Regulation of genetic variation in natural populations is a problem of primary importance to evolutionary biology. In the reported study, the repair efficiency of double strand DNA breaks was compared in six wild barley accessions from Israeli natural populations of H. spontaneum: three from mesic populations (one from Maalot and two from Mount Meron, Upper Galilee) and three from xeric populations (one from Wadi Quilt in the Judean Desert and two from Sede Boqer, in the northern Negev Desert). Pulsed field gel electrophoresis was used to score double-strand breaks of DNA (DSBs) caused by methyl methanesulphonate (MMS) treatment. All six accessions were also tested for heat tolerance: four of these, three xeric and one mesic (from Maalot population), were scored as heat tolerant whereas both accessions from Mount Meron population displayed heat sensitivity. MMS caused a significant increase in the level of DSBs relative to the control in all accessions. The major questions were whether and how the efficiency of DNA repair after mutagenic treatment is affected by the environmental conditions and accession’s adaptation to these conditions. Differences were found among the accessions in the repair pattern. Plants of two out of the four heat tolerant accessions did not manage to repair DNA neither at 25°C nor at 37°C. The remaining two heat tolerant accessions significantly repaired the breaks at 37°C, but not at 25°C. By contrast, plants of the two heat susceptible accessions significantly lowered the level of DSBs at 25°C but not at 37°C. Therefore, the accessions that proved capable to repair the induced damages in DNA at one of the two temperatures displayed a pattern that may imply the existence of a negative feedback mechanism in regulation of genetic variation. Such a dependence of DNA integrity on environment and genotype may serve an important factor for maintaining relatively high level of mutability without increasing the genetic load.  相似文献   

16.
Neoplastic cells frequently have an increased number of transferrin receptors. Coupling transferrin to an anti-neoplastic drug has the potential to overcome multidrug resistance (MDR). The purpose of this study was to examine the distribution and action of doxorubicin-transferrin conjugate (DOXTRF) in a leukaemia cell line (HL60), a multidrug-resistant leukaemia cell line (HL60ADR) and a normal tissue cell line (human fibroblasts). The intracellular accumulation of DOX and DOX-TRF was monitored by direct fluorescence. More DOX-TRF than free DOX was delivered to the tumour cells, and consecutively the levels of DNA double-strand breaks and apoptosis increased even in the multidrug-resistant cell line. In the normal tissue cell line, DOX-TRF did not accumulate, and therefore, the levels of DNA double-strand breaks and apoptosis did not increase. Cell viability was determined using the MTT assay. The IC50 for DOX-TRF was lower than the IC50 value for the free drug in both leukaemia cell lines. The IC50 values for the HL60 cells were 0.08 μM for DOX and 0.02 μM for DOX-TRF. The IC50 values for HL60ADR cells were 7 μM for DOX and 0.035 μM for DOX-TRF. In conclusion, DOX-TRF was able to overcome MDR in the leukaemia cell lines while having only a very limited effect on normal tissue cells.  相似文献   

17.
Using a semiquantitative PCR-based approach, a breakpoint cluster region of AML1 was associated with the nuclear matrix. Inhibition of topoisomerase II (topoII) by etoposide stimulated the appearance of histone γH2AX foci, indicative of DNA double-strand breaks (DSBs). The majority of these foci were associated with the nuclear matrix. Nuclear matrix-associated multiprotein complexes involved in topoII-induced DNA DSB repair were visualized. Colocalization studies demonstrated that these complexes included the main components of the nonhomologous end joining repair system (Ku80, DNA-PKcs, and DNA ligase IV). Thus, it was suggested that nonhomologous DNA end joining is a possible mechanism of topoII-induced chromosomal rear-rangements.  相似文献   

18.
    
Small tandem DNA duplications in the range of 15 to 300 base-pairs play an important role in the aetiology of human disease and contribute to genome diversity. Here, we discuss different proposed mechanisms for their occurrence and argue that this type of structural variation mainly results from mutagenic repair of chromosomal breaks. This hypothesis is supported by both bioinformatical analysis of insertions occurring in the genome of different species and disease alleles, as well as by CRISPR/Cas9-based experimental data from different model systems. Recent work points to fill-in synthesis at double-stranded DNA breaks with complementary sequences, regulated by end-joining mechanisms, to account for small tandem duplications. We will review the prevalence of small tandem duplications in the population, and we will speculate on the potential sources of DNA damage that could give rise to this mutational signature. With the development of novel algorithms to analyse sequencing data, small tandem duplications are now more frequently detected in the human genome and identified as oncogenic gain-of-function mutations. Understanding their origin could lead to optimized treatment regimens to prevent therapy-induced activation of oncogenes and might expose novel vulnerabilities in cancer.  相似文献   

19.
In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.  相似文献   

20.
It has been recently demonstrated that yeast cells are able to partially regress chromosome segregation in telophase as a response to DNA double-strand breaks (DSBs), likely to find a donor sequence for homology-directed repair (HDR). This regression challenges the traditional concept that establishes anaphase events as irreversible, hence opening a new field of research in cell biology. Here, the nature of this new behavior in yeast is summarized and the underlying mechanisms are speculated about. It is also discussed whether it can be reproduced in other eukaryotes. Overall, this work brings forwards the need of understanding how cells attempt to repair DSBs when transiting the latest stages of mitosis, i.e., anaphase and telophase.  相似文献   

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