The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human ...cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.
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•Resource of 31 genome-scale CRISPR screens against DNA-damaging agents•Cytotoxicity of G-quadruplex ligand pyridostatin involves TOP2 trapping•The bone-marrow failure syndrome gene ERCC6L2 codes for an NHEJ factor•The ELOF1 and STK19 proteins are candidate TC-NER factors
A set of CRISPR screens in cells treated with different genotoxic agents illuminates the cellular response to DNA damage, identifying new factors in several repair pathways and pinpointing a novel drug mechanism-of-action.
The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of ...resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identify a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.
53BP1 with its downstream proteins, RIF1, PTIP and REV7, antagonizes BRCA1-dependent homologous recombination (HR) and promotes non-homologous end joining (NHEJ) in an unclear manner. Here we show ...that REV7 forms a complex with two proteins, FAM35A and C20ORF196. We demonstrate that FAM35A preferentially binds single-strand DNA (ssDNA) in vitro, and is recruited to DSBs as a complex with C20ORF196 and REV7 downstream of RIF1 in vivo. Epistasis analysis shows that both proteins act in the same pathway as RIF1 in NHEJ. The defects in HR pathway to repair DSBs and the reduction in resection of broken DNA ends in BRCA1-mutant cells can be largely suppressed by inactivating FAM35A or C20ORF196, indicating that FAM35A and C20ORF196 prevent end resection in these cells. Together, our data identified a REV7-FAM35A-C20ORF196 complex that binds and protects broken DNA ends to promote the NHEJ pathway for DSB repair.
An aptamer based colorimetric assay is described for the determination of zearalenone (ZEN). It is based on the inhibition of the peroxidase-mimicking activity of gold nanoparticles (AuNPs) by the ...ZEN aptamer. However, in the presence of ZEN, the aptamer is bound by ZEN and can no longer inhibit the peroxidase-mimicking activity of AuNPs. The color change of solution is related to ZEN concentration and observed with bare eyes. Under optimal conditions, the absorbance (at 630 nm) increases linearly in the ZEN concentration range of 10–250 ng·mL
−1
, and the limit of detection is 10 ng·mL
−1
. The specificity of the assay was verified by studying the effect of potential interferents. The recoveries from ZEN spiked corn and corn oil range from 92 to 110%, and the relative standard deviations are between 2.4 and 6.4%. The results are in good agreement with those obtained by an ELISA.
Graphical abstract
Schematic presentation of colorimetric assay for rapid and sensitive determination of zearalenone (ZEN) based on the inhibition of ZEN aptamer on the the peroxidase-like activity of gold nanoparticle (AuNPs).
Iris recognition and favor because of its high recognition rate, noninvasive and simple algorithm and other advantages, in a variety of biometric identification technology is very prominent. The iris ...texture feature extraction is the core of the iris recognition algorithm. Fractal geometry theory provides new ideas and methods to express nonlinear image information, the fractal dimension is an important parameter of fractal geometry, is a measure of complexity of irregular change, covering blanket dimension can better reflect the graphics changes in different resolution characteristics; missing is the fractal dimension and independent statistics, is a supplement to the fractal dimension, overcome the different texture characteristics may have the same fractal dimension of the problem. This paper presents a blanket and missing items based on the combination of texture feature extraction algorithm, can make full use of radiation in different resolution iris texture information and texture, classification ability make feature matrix has a better. Iris matching is the key of iris recognition. How to effectively carry out the matching of the iris code matrix is the decisive step in iris recognition. In this paper based on the normalized correlation classifier, the matching method of cyclic shift, eliminated from the same eyes of different iris image due to differences in rotation caused, improve matching accuracy.
The replication protein A (RPA) complex binds single-stranded DNA generated at stalled replication forks and recruits other DNA repair proteins to promote recovery of these forks. Here, we identify ...Ewing tumor-associated antigen 1 (ETAA1), which has been linked to susceptibility to pancreatic cancer, as a new repair protein that is recruited to stalled forks by RPA. We demonstrate that ETAA1 interacts with RPA through two regions, each of which resembles two previously identified RPA-binding domains, RPA70N-binding motif and RPA32C-binding motif, respectively. In response to replication stress, ETAA1 is recruited to stalled forks where it colocalizes with RPA, and this recruitment is diminished when RPA is depleted. Notably, inactivation of the ETAA1 gene increases the collapse level of the stalled replication forks and decreases the recovery efficiency of these forks. Moreover, epistasis analysis shows that ETAA1 stabilizes stalled replication forks in an ataxia telangiectasia and Rad3-related protein (ATR)-independent manner. Thus, our results reveal that ETAA1 is a novel RPA-interacting protein that promotes restart of stalled replication forks.
To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook ...flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.
The replication protein A (RPA) complex binds single-stranded DNA generated at stalled replication forks and recruits other DNA repair proteins to promote recovery of these forks. Here, we identify ...Ewing tumor-associated antigen 1 (ETAA1), which has been linked to susceptibility to pancreatic cancer, as a new repair protein that is recruited to stalled forks by RPA. We demonstrate that ETAA1 interacts with RPA through two regions, each of which resembles two previously identified RPA-binding domains, RPA70N-binding motif and RPA32C-binding motif, respectively. In response to replication stress, ETAA1 is recruited to stalled forks where it colocalizes with RPA, and this recruitment is diminished when RPA is depleted. Notably, inactivation of the ETAA1 gene increases the collapse level of the stalled replication forks and decreases the recovery efficiency of these forks. Moreover, epistasis analysis shows that ETAA1 stabilizes stalled replication forks in an ataxia telangiectasia and Rad3-related protein (ATR)-independent manner. Thus, our results reveal that ETAA1 is a novel RPA-interacting protein that promotes restart of stalled replication forks.
The slump of coal mining's roof is one of the mine's great calamities in underground exploitation, the influence factors of the roof unsteadiness is rather complex. The essence of crack evolution is ...rock transform. Although the crack of rock are different between each other, from the viewpoint of fracture mechanics, the crack creating always include 3 stages that turn into and grow slowly, expand and rupture instantly, at the same time, every stage of transform leaves a lot of transmute traces in the crack. Through observe experiment, the relationship between stress and crack can be obtained, but having no crack's actual width. This paper applies the wavelet analysis to identify the image, which reflects the act.ual position of the inside crack. Though the CT, the image ridge can be checked out, and the relationship between the ridge and actual crack can be defined by the linear-return method.
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