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Objective:To obtain the relative biological effectiveness (RBE) of DNA double strand breaks (DSB) clusters by tracing the mechanism of radiated DNA damage, and explore the relationship among the biological effectiveness of DNA damage, chromosomal aberrations and germ cell death.Methods:Taking low-energy electrons, protons, and α particles as the research objects, this study simulated the process that cell nuclei were exposed to particle radiation using a radiation-related physicochemical model. On the ground of the DSB density-based spatial clustering of applications with noise (DBSCAN) algorithm, the DSB cluster classification method was improved to weaken the connection between the DSBs and the random distribution assumptions of energy depositions during the simulation. In this manner, the DSB clusters can be much closer to a non-random distribution. Furthermore, this study obtained the yields of DSB clusters and proposed a method to calculate the RBE values of DSB clusters.Results:The calculated RBE value (12.29) of DSB clusters of 2 MeV α particles was similar to the experimental RBE values of chromosomal fragments (15.3±5.9) and cell survival (14.7±5.1).Conclusions:After high-LET ionizing radiation, unlike the single DSB, the RBE of DSB clusters was similar to that of chromosomal aberration and cell survival.
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DNA double-strand break(DSB) is a serious form of DNA damage in cells, which is closely related to a variety of genomic instability diseases, including cancer, abnormal recombination and neuronal development. Due to the limitations of cost and technical threshold, high-resolution DSB mapping by high-throughput sequencing technology is very limited. This hinders our understanding of the DSB situation in the genomes of different species. Therefore, we developed a classification prediction model based on random Forest(RF), support vector machine(SVM) and logistic regression(LR) classifiers to predict DSB loci in the whole genome of human NHEK cells. In addition to the epigenetic features and DNA shape features commonly used in previous prediction studies, we found that DNA sequence features(kmer frequency, GC content, GC-skew, Mutual Information) can also characterize DSB sites. At the same time, the prediction accuracy is improved after considering DNA physical properties, chemical shifts and autocorrelation information. After combining all the above features, logistic regression(LR) has the best prediction performance(AUC = 0. 97), which is comparable to previous prediction(AUC = 0. 964). In addition, the optimal feature collection consisting of 294 features was obtained by the incremental feature search method, and the corresponding AUC value reached 0. 974.
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@#Gene editing tools with nucleases as the main component have now implemented programmable targeted mutagenesis or insertion or deletion of mammalian genomes.From zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas system to safer and more accurate Cas9 fusion protein gene editing tools and other nuclease gene editing tools, this paper systematically describes the development and evolution of gene editing, with detailed introduction to the development and optimization of next-generation gene editing tools, and a prospect of the clinical application of and challenges for gene editing tools.
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Objective:To investigate the regulating molecules and acting mechanism of TAB182 in HR pathway.Methods:TAB182 in human breast cancer MCF-7 cells was knocked down by shRNA strategy, the TAB182 knockdown MCF-7 as the TAB182 knockdown group, and the MCF-7 cell using the shRNA negative control as the TAB182 negative control group. RNA sequencing and qRT-PCR were performed to screen and verify the differentially expressed genes of HR pathway related to TAB182 depression. Western blot was used to detect protein expression. Immunofluorescence staining of nuclear RAD51 and BrdU was used to check the 3′ ssDNA formation by the end resection. The cell cycle arrest and apoptosis were measured by flow cytometry. Cloning formation assay was used to evaluate the sensitivity TAB182-knockdown cells to radiation.Results:Both quantitative RNA sequencing and qRT-PCR assays showed that TAB182-knockdown significantly decreased the mRNA expression of RPA2( t=17.97, P<0.05). Compared with the TAB182 negative control group, the protein level of RPA2, the number of RAD51 foci, and the 3′ ssDNA-binding nuclear protein marker BrdU in TAB182-knockdown cells were significantly reduced. At 4, 8, and 12 h after actinomycin D treatment, the attenuation of RPA2 mRNA in the TAB182-knockdown cells was accelerated ( t=5.37, 3.79, 3.69, P<0.05). Compared with the TAB182 negative control group, the radiosensitivity and radiation-induced apoptosis in the TAB182-knockdown group were increased ( t=3.48, 11.05, P<0.05), and at 24 h after irradiation, the cell cycle block time was prolonged ( t=8.40, P<0.01). Conclusions:TAB182 plays a role in maintaining RPA2 mRNA stability, thereby promoting HR repair. TAB182 knockdown cells are highly sensitive to ionizing radiation.
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DNA repair enzymes are proteins that detect and repair physical damage to DNA induced by radiation, ultraviolet light, or reactive oxygen species. The repair of DNA damage prevents the loss of genetic information, the creation of double-strand breaks, and the formation of DNA crosslinks. The time-dependent reduction of functional properties is known as aging. Mitochondrial malfunction and the buildup of genetic damage are two common factors of aging. In fact, the poor maintenance of nuclear and mitochondrial DNA is likely a major factor in aging. When the DNA repair machinery isn't operating fine, DNA lesions and mutations can occur, which can lead to cancer development. In fact, the poor maintenance of nuclear and mitochondrial DNA is likely a major factor in aging. When the DNA repair enzymes isn't operating fine, DNA lesions and mutations can occur, which can lead to cancer development. The large number of alterations per cell, which can reach 105, has been identified as a driving mechanism in oncogenesis. These findings show that abnormalities in the DNA repair pathway contribute to the senescence as well as cancer. Nucleotide excision repair (NER), base excision repair (BER), double-strand break repair, mismatch repair (MMR), are all major DNA repair processes in mammalian cells. BER excises mostly oxidative and alkylation DNA damage, NER removes bulky, helix-distorting lesions from DNA (e.g., ultraviolet (UV) photodimers), MMR corrects replication errors
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Aim To explore the inhibitory effect of the on gastric cancer cells from the perspective of DNA total terpenoids of Celastrus orbiculatus Thunb (TTC) damage response. Methods CCK-8 experiment was conducted to investigate the toxic effects of different concentrations
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Meiosis is an essential step in gametogenesis which is the key process in sexually reproducing organisms as meiotic aberrations may result in infertility. In meiosis, programmed DNA double-strand break (DSB) formation is one of the fundamental processes that are essential for maintaining homolog interactions and correcting segregation of chromosomes. Although the number and distribution of meiotic DSBs are tightly regulated, still abnormalities in DSB formation are known to cause meiotic arrest and infertility. This review is a detailed account of molecular bases of meiotic DSB formation, its evolutionary conservation, and variations in different species. We further reviewed the mutations of DSB formation genes in association with human infertility and also proposed the future directions and strategies about the study of meiotic DSB formation.
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Humanos , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Infertilidad/genética , Meiosis/fisiologíaRESUMEN
Objective:To clarify the role of classic Wnt signaling pathway in the radioresistance of esophageal cancer cells (ECC), and investigate the underlying mechanism, aiming to identify critical molecular targets for clinically enhancing the radiosensitivity of esophageal cancer.Methods:The radiosensitivity of four types of ECCs (EC9706, ECA109, KYSE70 and KYSE150) were assessed by colony formation assay. Western blot and RT-PCR were used to detect the activation of classical Wnt signaling pathway after irradiation. Classic Wnt signaling pathway activator (AZD2858) and inhibitor (XAV-939) were utilized to comprehensively evaluate the effect of classic Wnt signaling pathway on the radiosensitivity of ECCs. Cellular immunofluorescence staining was performed to detect the production and repair of DNA double-strand breaks (DSB), as well as the foci formation of DSB repair proteins after irradiation.Results:The results of colony formation assay showed that the radiosensitivity of four types of ECCs from high to low was EC9706, ECA109, KYSE70 and KYSE150. In KYSE150, a radioresistant cell type, the level of nuclear β-catenin and the transcription of c-Myc gene were significantly increased after irradiation (both P<0.05). However, in EC9706, a radiosensitive cell type, the level of nuclear β-catenin and c-Myc gene transcription were not affected by irradiation (both P>0.05). Moreover, EC9706 cells showed enhanced radioresistance in the presence of AZD2858( P<0.05), whereas XAV-939 treatment decreased the radioresistance in KYSE150 cells ( P<0.05). AZD2858 accelerated the DSB repair in EC9706 cells ( P<0.05), whereas XAV-939 delayed the DSB repair in KYSE150 cells ( P<0.05). Furthermore, the results of immunofluorescence staining showed that XAV-939 reduced the DSB repair capacity by inhibiting homologous recombination repair-related proteins (BRCA1 and RAD51) rather than non-homologous end junction repair-related proteins (Ku80 and XRCC4). Conclusions:The classic Wnt signaling pathway participates in the regulation of radiosensitivity in ECCs by regulating the homologous recombination repair of DSB after irradiation. Inhibition of the classic Wnt signaling pathway can counteract the radioresistance of ECCs and enhance the killing effect of irradiation on ECCs.
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Objective:To construct a random forest classification model of DNA double strand breaks (DSB) induced by ionizing radiation and investigate the genome-wide distribution of DSB.Methods:The GRCh38 reference genome was divided into 50 kilobase fragments. Then these genomic fragments were separated into low-level or high-level regions of ionizing radiation-induced DSB according to the sequencing data of MCF-7 cells. The data of eight epigenetic features were used as input. Two thirds of the data were randomly assigned to the training set, and the rest of the data was assigned to the test set. A random forest classification model with 100 decision trees was constructed. The importance of epigenetic features in the classification model was analyzed and displayed.Results:The accuracy score of the random forest classification model on the test set was 99.4%, the precision score was 98.9% and the recall score was 99.9%. The area under the receiver operating characteristic curve was 0.994. Among the eight epigenetic features, H3K36me3 and DNase markers were the most important variables. The enrichments of the two markers in DSB high-level regions were much higher than those in DSB low-level regions.Conclusions:The random forest classification model could precisely predict the genome-wide levels of DSB induced by ionizing radiation in the 50 kilobase window based on epigenetic features. Analysis revealed that these DSB might primarily distribute in the actively transcribed sites in the genome.
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@#Objective To explore the effects and molecular mechanisms of histone methylase G9a inhibitor BIX-01294 on apoptosis in esophageal squamous cell carcinoma (ESCC). Methods MTT assay and Colony-forming Units were adopted to determine the effects of BIX-01294 on the growth and proliferation of ESCC cell lines EC109 and KYSE150. Flow cytometry was used to analyze the apoptosis status of ESCC cells after the treatment of BIX-01294. The effects of BIX-01294 treatment on the expressions of G9a catalytic product H3K9me2, DNA double-strand break (DSB) markers, and apoptosis-related proteins were detected by Western blotting. Results BIX-01294 inhibited the growth of EC109 and KYSE150 cells in a dose-dependent manner (P<0.05), and BIX-01294 with the inhibitory concentration 50%(IC50) significantly inhibited the formation of colony (P<0.05). After 24 hours treatment of BIX-01294 (IC50), the apoptosis rate of EC109 cells increased from 11.5%±2.1% to 42.5%±5.4%, and KYSE150 cells from 7.5%±0.9% to 49.2%±5.2%(P<0.05). The expression level of the G9a catalytic product, H3K9me2, significantly decreased (P<0.05); while the expression of the DSB marker γH2AX was dramatically enhanced (P<0.05). We also found that the mitochondrial apoptosis pathway was activated and the expression levels of cleaved caspase3 and cleaved PARP were significantly elevated (P<0.05). Conclusion BIX-01294, the inhibitor of methyltransferase G9a, prompted apoptosis in ESCC cells by inducing DSB damage and activating mitochondrial apoptosis pathway.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.
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DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks (DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair. Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes (DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.
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Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break (DSB) signaling. P53-binding protein 1 (53BP1) plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining (NHEJ)-mediated DSB repair pathway that rejoins DSB ends. New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination (HR) signaling. This review focuses on the up- and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair, which in turn promotes the sensitivity of poly(ADP-ribose) polymerase inhibitor (PARPi) in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies.
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La función original de los sistemas CRISPR/Cas es destruir el DNA de virus bacterianos. Este sistema ha evolucionado para identificar y cortar secuencias de diferentes DNA de virus de DNA evitando la infección. En la célula, está compuesto de genes Cas que producen nucleasas guiadas por RNA capaces de cortar el DNA. Si el RNA guía encuentra DNA de un virus con el que se puede emparejar, recluta a la nucleasa Cas9 que lo corta. Este sistema es utilizado in vitro para editar genes basándose en la producción de rupturas de doble cadena y su posterior reparación. Actualmente existen varias plataformas para el diseño de RNAs guía, aunque también es posible realizarlo de forma manual. Los componentes del sistema son entregados a la célula mediante un plásmido o una ribonucleoproteína. En esta revisión nos centraremos en la función original de CRISPR/Cas en procariotas y en cómo los investigadores la han modificado para proporcionar nuevas técnicas de edición de genomas. Discutiremos sobre las ventajas de esta nueva técnica, las formas en que podemos utilizarla y algunas de las limitaciones que aún encontramos en su aplicación
The original function of CRISPR/Cas systems is to destroy the DNA of bacterial viruses. This system has evolved to identify sequences of different DNA viruses and cut them in order to avoid infection. In the cell, the system is made up of Cas genes which produce RNA-guided nucleases capable of cutting DNA. If the guide RNA finds viral DNA with which it can pair up, it recruits the Cas9 nuclease to cut it. This system is used in vitro for gene edition, relying on the production of double-strand breaks and their subsequent repair. Currently, there are several platforms for the design of the guide RNA, and it is also possible to design it manually. The components of the system can be delivered to the cell through a plasmid or through a ribonucleoprotein. In this review we will focus on the original function of CRISPR/Cas in prokaryotes, and in how researchers have modified it in order to provide new genome editing techniques. We will discuss the advantages of this new technique, the ways in which it can be used, and some of the limitations found in its application
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Sistemas CRISPR-Cas , Edición Génica , ADNRESUMEN
Applying poly(ADP-ribose) polymerase inhibitors (PARPi) to the treatment of cancers with homologous recombination deficiency (HRDness) has been a great advance in the field of molecular therapeutics. However, in the clinic patients lacking the specific mutations or developing reverse mutations in the process of PARPi treatment may not benefit from PARPi monotherapy. Therefore, targeting homologous recombination (HR) repair with molecularly targeted agents is becoming an attractive research focus and is raising the concept of "chemical HRDness". HR repair is an evolutionarily conserved and extensively regulated process that employs sister chromatids as the template to repair DNA double-strand breaks with high fidelity. In addition to directly targeting HR components, modulation of regulatory pathways controlling HR repair is effective in achieving the "HRDness" phenotype; this includes modulation of the cell cycle checkpoint regulatory pathway, the phosphatidylinositol 3-kinase (PI3K) signaling pathway, the chromatin remodeling pathway, etc. Targeting HR repair can not only result in "synthetic lethality" when combined with PARPi, but also sensitizes cancers to traditional radio/chemotherapy and novel immunotherapy. In this review we describe the HR repair pathway and its regulatory pathways, summarize the preclinical and clinical outcomes of targeting HR repair, discuss the remaining problems in this field and provide a prospective on its application in tumor therapy.
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Recent research has shown that cell-free chromatin (cfCh) particles that are released from the billions of cells that die in thebody everyday can enter into healthy cells, integrate into their genomes and induce dsDNA breaks and apoptotic responses.Genomic integration of cfCh activates NFjB suggesting a novel mechanism of induction of systemic inflammation. SinceDNA damage and inflammation are underlying pathologies in multiple devastating acute and chronic disease conditions,the discovery of agents that can inactivate cfCh may provide therapeutic possibilities.
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The small ubiquitin-like modified protein (SUMO) is a protein structurally similar to ubiquitin which is involved in post-translational modification of proteins. SUMOylation refers to the process that SUMO molecule covalently binding to the specific lysine site of target proteins through maturation, activation, binding and ligation by ubiquitin-like specific protease 1 (Ulp1), E1 activating enzyme, E2 binding enzyme, and E3 ligase. SUMOylation alters the activity of target proteins, which is involved in the regulation of various cellular functions such as transcriptional regulation, regulation of embryonic development, cellular stress, maintenance of chromatin structure and genomic stability. In recent years, it has been found that SUMOylation modification is also widely involved in DNA damage repair, especially DNA double-strand breaks (DSBs), which are the most serious types of DNA damage. SUMOylation is involved in almost all processes of DSBs repair, so its role in DNA damage repair has become a research hotspot. In this paper, the research progress of the regulation of SUMOylation in DSBs repair was reviewed.
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Objective To investigate the expressions of phosphorylated H2AX (γH2AX) and p53-binding protein 1 (53BP1) in DNA oxidative damage of human bronchial epithelial (HBE) cells.Methods The HBE cells were treated with 0,25,50,100,200,400 μmol/L of hydrogen peroxide (H2O2) for 1 hour,respectively,and their DNA oxidative damages displaying as double-strand breaks (DSBs) were induced.The viability and apoptosis of HBE cells were measured by the CCK-8 method and flow cytometry,respectively.The expression status of γH2AX and 53BP1 in nucleus of HBE cells was observed by a fluorescence microscope.The expression levels of γH2AX,53BP1 and BRCA1 were determined by western blot.Results Compared with the control (0 μmol/L of H2O2),the via bility of HBE cells treated with 25 μmol/L of H2O2 (1.07 ±0.01) increased,while those with 50,100,200,400 μmol/L of H2O2 (0.97 ± 0.01,0.96 ± 0.01,0.95 ± 0.01,0.94 ± 0.01) decreased significantly (F =50.35,P ＜ 0.01).The apoptosis rates of HBE cells treated with 50,100,200,400 μ mol/L of H2O2 ([7.54 ± 0.57] ％,[7.84 ± 0.68] ％,[8.40 ± 0.50] ％ and [14.03 ± 1.03] ％) were significantly higher than that with 0 μmol/L of H2O2 ([4.65 ± 0.32] ％,F =35.879,P ＜ 0.01).Compared with the control (0 μmol/L of H2O2),the average fluorescence intensity of γH2AX in nucleus of HBE cells treated with 25,50,100,200,400 μmol/L of H2O2 increased significantly (F =223.97,P ＜ 0.01),while those of 53BP1 in nucleus of HBE cells treated with 50,100,200,400 μmol/L of H2O2 decreased significantly (F =117.78,P ＜ 0.01).The results of western blot showed that the expres sion levels of γH2AX increased with the increase of H2O2 concentration,while that of 53BP1 and BRCA1 was on the contrary (F =96.20,21.92 and 11.55,respectively,P ＜0.01).Conclusion In the oxidative damage of HBE cells induced by H2O2,γH2AX may be used as a marker of DNA oxidative damage,while the decreased expression of 53BP1 suggests that other mechanisms to repair the DNA damage sites may exist.
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Objective To investigate the expression of bone marrow γ-H2AX in the patients with multiple myeloma(MM) and its correlation with the prognosis.Methods The patients with newly diagnosed MM in this hospital were selected as the case group,and the patients with non-hemopoietic system tumor without obvious morphological abnormalities by bone marrow smear and biopsy served as the control group.The immunohistochemistry was adopted to detect the expression level of bone marrow γ-H2AX in the cases group and control group,the image-Pro Plus(IPP) semiquantitative analysis was performed.The expression differences were compared between the two groups,moreover the case group was re-divided into the strong expression group and weak expression group according to γ-H2AX expression level.Then the relation ship between γ-H2AX expression level and the prognosis in the patients with MM.Results The bone marrow γ-H2AX expression level in the case group was significantly higher than that in the control group (P＜0.05);the level of γ-H2AX expression in the strong expression group was significantly stronger than that in the weak expression group (P＜0.05).Conclusion The level of γ-H2AX expression was higher among MM patients,and the over expression of γ-H2AX predicts the shorter survival time.
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The stability of cell genetic material is influenced by a variety of factors, both internal and external, which can cause various types of DNA damage, such as DNA alkylation, oxidation, mismatching, loop structure, atypical DNA structure, single-strand break, and double-strand break.These DNA damages disrupt cellular homeostasis and dynamic equilibrium, which cause gene mutations, chromosomal abnormalities, and even degradation, aging, and death at different biological levels.By searching and identifying DNA damage sites, the cell activates a series of biochemical pathways, coordinates the progress of DNA replication and transcription, and then repairs the DNA damage.In this way, the cell maintains its independence and stability.While radiotherapy plays a role in eliminating tumors by DNA damages, it also initiates DNA damage responses.Among the responses, base excision repair, nucleotide excision repair, mismatch repair, double-strand break repair, and post-translesion synthesis repair play a key role in repairing the damages.The dysfunction of these repair pathways will cause differences in tumor radiation sensitivity.This paper summarizes recent research results in DNA damage repair, and focuses on the types of DNA damage and their repair mechanisms, so as to promote the understanding of the great significance of this field and to provide a theoretical basis for exploring the application of DNA damage repair pathways in tumor therapy.