Non-homologous End-joining Pathway Research Articles

KuINins as a New Class of HIV-1 Inhibitors That Block Post-Integration DNA Repair.

Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins.

The MRE11–ATM–SOG1 DNA damage signaling pathway confers rice immunity to Xanthomonas oryzae

Plants are constantly exposed to microbial pathogens in the environment. One branch of innate plant immunity is mediated by cell-membrane-localized receptors, but less is known about associations between DNA damage and plant immune responses. Here, we show that rice (Oryza sativa) mesophyll cells are prone to DNA double-stranded breaks (DSBs) in response to ZJ173, a strain of Xanthomonas oryzae pv. oryzae (Xoo). The DSB signal transducer ataxia telangiectasia mutated (ATM), but not the ATM and Rad3-related branch, confers resistance against Xoo. Mechanistically, the MRE11–ATM module phosphorylates suppressor of gamma response 1 (SOG1), which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection. Intriguingly, overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining (NHEJ) pathway to the alternative NHEJ and homologous recombination pathways at Xoo-induced DSBs. The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines. Overall, the MRE11–ATM–SOG1 pathway serves as a prime example of plant–pathogen interactions that occur via host non-specific recognition. The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.

Evaluation of topotecan and 10-hydroxycamptothecin on Toxoplasma gondii: Implications on baseline DNA damage and repair efficiency

Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes toxoplasmosis in humans and animals worldwide. Despite its prevalence, there is currently no effective vaccine or treatment for chronic infection. Although there are therapies against the acute stage, prolonged use is toxic and poorly tolerated. This study aims to explore the potential of repurposing topotecan and 10-hydroxycamptothecin (HCPT) as drugs producing double strand breaks (DSBs) in T. gondii. DSBs are mainly repaired by Homologous Recombination Repair (HRR) and Non-Homologous End Joining (NHEJ). Two T. gondii strains, RHΔHXGPRT and RHΔKU80, were used to compare the drug's effects on parasites. RHΔHXGPRT parasites may use both HRR and NHEJ pathways but RHΔKU80 lacks the KU80 protein needed for NHEJ, leaving only the HRR pathway. Here we demonstrate that topotecan and HCPT, both topoisomerase I venoms, affected parasite replication in a concentration-dependent manner. Moreover, variations in fluorescence intensity measurements for the H2A.X phosphorylation mark (γH2A.X), an indicator of DNA damage, were observed in intracellular parasites under drug treatment conditions. Interestingly, intracellular replicative parasites without drug treatment show a strong positive staining for γH2A.X, suggesting inherent DNA damage. Extracellular (non-replicating) parasites did not exhibit γH2A.X staining, indicating that the basal level of DNA damage is likely to be associated with replicative stress. A high rate of DNA replication stress possibly prompted the evolution of an efficient repair machinery in the parasite, making it an attractive target. Our findings show that topoisomerase 1 venoms are effective antiparasitics blocking T. gondii replication.

A CRISPR-drug perturbational map for identifying compounds to combine with commonly used chemotherapeutics

Combination chemotherapy is crucial for successfully treating cancer. However, the enormous number of possible drug combinations means discovering safe and effective combinations remains a significant challenge. To improve this process, we conduct large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritize neuroblastoma, the most common extracranial pediatric solid tumor, where ~50% of high-risk patients do not survive. Our screen examines all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest existing drug combination screens. Using dense drug-drug rescreening, we find that the top CRISPR-nominated drug combinations are more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discover that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using patient-derived xenograft (PDX) models. Our findings provide a valuable resource and demonstrate the feasibility of using targeted CRISPR knockout to discover combinations with common chemotherapeutics, a methodology with application across all cancers.

Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma.

Toxoplasma gondii is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome-the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 mya, revealed that the movement and fixation of five NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb), and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together, these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.

MiR-223-3p promotes genomic stability of hematopoietic progenitors after radiation

When hematopoietic cells are overwhelmed with ionizing radiation (IR) DNA damage, the alternative non-homologous end-joining (aNHEJ) repair pathway is activated to repair stressed replication forks. While aNHEJ can rescue cells overwhelmed with DNA damage, it can also mediate chromosomal deletions and fusions, which can cause mis-segregation in mitosis and resultant aneuploidy. We previously reported that a hematopoietic microRNA, miR-223-3p, normally represses aNHEJ. We found that miR-223-/- mice have increased survival of hematopoietic stem and progenitor cells (HSPCs) after sublethal IR. However, this came at the cost of significantly more genomic aberrancies, with miR-223-/- hematopoietic progenitors having increased metaphase aberrancies, including chromothripsis, and increased sequence abnormalities, especially deletions, which is consistent with aNHEJ. These data imply that when an HSPC is faced with substantial DNA damage, it may trade genomic damage for its own survival by choosing the aNHEJ repair pathway, and this choice is regulated in part by miR-223-3p.

AZD-7648, a DNA-PK Inhibitor, Induces DNA Damage, Apoptosis, and Cell Cycle Arrest in Chronic and Acute Myeloid Leukemia Cells.

The non-homologous end joining pathway is vital for repairing DNA double-strand breaks (DSB), with DNA-dependent protein kinase (DNA-PK) playing a critical role. Altered DNA damage response (DDR) in chronic (CML) and acute myeloid leukemia (AML) offers potential therapeutic opportunities. We studied the therapeutic potential of AZD-7648 (DNA-PK inhibitor) in CML and AML cell lines. This study used two CML (K-562 and LAMA-84) and five AML (HEL, HL-60, KG-1, NB-4, and THP-1) cell lines. DDR gene mutations were obtained from the COSMIC database. The copy number and methylation profile were evaluated using MS-MLPA and DDR genes, and telomere length using qPCR. p53 protein expression was assessed using Western Blot, chromosomal damage through cytokinesis-block micronucleus assay, and γH2AX levels and DSB repair kinetics using flow cytometry. Cell density and viability were analyzed using trypan blue assay after treatment with AZD-7648 in concentrations ranging from 10 to 200 µM. Cell death, cell cycle distribution, and cell proliferation rate were assessed using flow cytometry. The cells displayed different DNA baseline damage, DDR gene expressions, mutations, genetic/epigenetic changes, and p53 expression. Only HEL cells displayed inefficient DSB repair. The LAMA-84, HEL, and KG-1 cells were the most sensitive to AZD-7648, whereas HL-60 and K-562 showed a lower effect on density and viability. Besides the reduction in cell proliferation, AZD-7648 induced apoptosis, cell cycle arrest, and DNA damage. In conclusion, these results suggest that AZD-7648 holds promise as a potential therapy for myeloid leukemias, however, with variations in drug sensitivity among tested cell lines, thus supporting further investigation to identify the specific factors influencing sensitivity to this DNA-PK inhibitor.

Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic.

DNA damage and repair: underlying mechanisms leading to microcephaly.

DNA-damaging agents and endogenous DNA damage constantly harm genome integrity. Under genotoxic stress conditions, the DNA damage response (DDR) machinery is crucial in repairing lesions and preventing mutations in the basic structure of the DNA. Different repair pathways are implicated in the resolution of such lesions. For instance, the non-homologous DNA end joining and homologous recombination pathways are central cellular mechanisms by which eukaryotic cells maintain genome integrity. However, defects in these pathways are often associated with neurological disorders, indicating the pivotal role of DDR in normal brain development. Moreover, the brain is the most sensitive organ affected by DNA-damaging agents compared to other tissues during the prenatal period. The accumulation of lesions is believed to induce cell death, reduce proliferation and premature differentiation of neural stem and progenitor cells, and reduce brain size (microcephaly). Microcephaly is mainly caused by genetic mutations, especially genes encoding proteins involved in centrosomes and DNA repair pathways. However, it can also be induced by exposure to ionizing radiation and intrauterine infections such as the Zika virus. This review explains mammalian cortical development and the major DNA repair pathways that may lead to microcephaly when impaired. Next, we discuss the mechanisms and possible exposures leading to DNA damage and p53 hyperactivation culminating in microcephaly.

Xrcc5/KU80 is not required for the survival or activation of prophase-arrested oocytes in primordial follicles.

The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources. Even a single DNA double-strand break (DSB) can trigger oocyte apoptosis, which can lead to accelerated depletion of the ovarian reserve, early loss of fertility and menopause. Therefore, repair of DNA damage is important for preserving the quality of oocytes to sustain fertility across the reproductive lifespan. This study aimed to evaluate the role of KU80 (encoded by the XRCC5 gene) - an essential component of the non-homologous end joining (NHEJ) pathway - in the repair of oocyte DNA DSBs during reproductive ageing, and following insult caused by the DNA-damaging chemotherapies cyclophosphamide and cisplatin. To investigate the importance of KU80 following endogenous and exogenous DNA damage, ovaries from conditional oocyte-specific Xrcc5 knockout (Xrcc5 cKO) and wildtype (WT) mice that were aged or exposed to DNA damage-inducing chemotherapy were compared. Ovarian follicles and oocytes were quantified, morphologically assessed and analysed via immunohistochemistry for markers of DNA damage and apoptosis. In addition, chemotherapy exposed mice were superovulated, and the numbers and quality of mature metaphase- II (MII) oocytes were assessed. The number of healthy follicles, atretic (dying) follicles, and corpora lutea were similar in Xrcc5 cKO and WT mice at PN50, PN200 and PN300. Additionally, primordial follicle number and ovulation rates were similar in young adult Xrcc5 cKO and WT mice following treatment with cyclophosphamide (75mg/kg), cisplatin (4mg/kg), or vehicle control (saline). Furthermore, KU80 was not essential for the repair of exogenously induced DNA damage in primordial follicle oocytes. These data indicate that KU80 is not required for maintenance of the ovarian reserve, follicle development, or ovulation during maternal ageing. Similarly, this study also indicates that KU80 is not required for the repair of exogenously induced DSBs in the prophase-arrested oocytes of primordial follicles.

Design and Synthesis of Ketenimine Sulfonamide Conjugates through Multicomponent Reactions; A Combined Cytotoxic Analysis and Computational Exploration.

Multicomponent reactions involving zwitterion generated from dimethyl acetylenedicarboxylate, aryl sulfonamide, and isocyanide to generate sulfonamide-conjugated ketenimines is reported. The synthetic strategy adopted is highly atom economical and stereoselective. Ketenimine sulfonamide analogues are key intermediates for further synthetic conversions to generate a combinatorial library of compounds. Furthermore, sulfonamide compounds are known to possess a broad spectrum of biological applications. All the novel molecules synthesized exhibit the potential to target the nonhomologous DNA end-joining (NHEJ) pathway with cytotoxic ability. Computational studies compliment the in vitro biological assays of the 8 small-molecule inhibitors. DNA double-strand breaks (DSBs) are considered as the most lethal among different DNA damages. NHEJ repairs about 70% of the DSBs generated in cells within mammals. The DNA-dependent protein kinase catalytic subunit is one of the PI3 kinases associated with NHEJ. Compounds DK01-DK08 were investigated for their ability to induce cancer cell death by treating with two leukemic cell lines where NHEJ is high. Results showed that bromoaryl (DK04)- and nitroaryl (DK05)-conjugated molecules showed excellent biological activity, having IC50 values of ∼2 μM in Nalm6 cell lines.

Characterization of DNA Damage Response-Associated Somatic Mutations in Borderline Resectable and Locally Advanced Pancreatic Cancer

The role of radiation for pancreatic cancer remains controversial, with recent studies showing conflicting results, highlighting the need to develop biomarkers of radiation response. Despite its potential utility in predicting radiosensitivity, the landscape of somatic mutations in borderline resectable pancreatic cancer (BRPC) and locally advanced pancreatic cancer (LAPC), as related to DNA damage response (DDR), has not been well characterized. This study aimed to characterize the frequency of such mutations in a cohort of patients with BRPC/LAPC treated with neoadjuvant chemotherapy and stereotactic body radiotherapy (SBRT). Mutational data was collected from patients with BRPC/LAPC treated at a single institution with neoadjuvant chemotherapy and SBRT, followed by surgical resection from 2016-2021. Chemotherapy consisted of modified FOLFIRINOX or gemcitabine/nab-paclitaxel, and patients were treated with SBRT in 33 Gy in 5 fractions. Genomic data was obtained from either endoscopic biopsy or surgical specimens, and next-generation sequencing was performed either in-house with a Solid Tumor Panel or with FoundationOne CDx. Specific emphasis was placed on the characterization of double-strand DNA break (DSB) repair genes, as this is the type of tumor cell damage traditionally induced by radiation therapy. Genes associated with the two main pathways of DSB repair, non-homologous end joining (NHEJ) and homologous repair (HR), were analyzed. Specific HR pathway mutations assessed were BLM, BRCA1/2, MRE11, NBN, PALB2, RAD50, RAD51B-D, and RAD54L, while PRKDC mutations were assessed for the NHEJ pathway. Mutations in ATM, an important initiator of DDR pathways, were also analyzed. Additionally, the frequency of mutations in TP53, CDKN2A and SMAD4 in patients with concomitant KRAS mutations was assessed. Eighty-five patients were included in the study. Five (5.9%) patients had mutations in the NHEJ pathway of the PRKDC gene. Twenty (23.5%) patients had mutations in the HR pathway, including BRCA2 (10/85; 11.8%), PALB2 (5/85; 5.9%), BRCA1 (3/85; 3.5%), and RAD50 (1/85; 1.2%). Six (7.1%) patients had mutations in ATM. No patients were found to have mutations in BLM, RAD51B-D, RAD54L, or NBN. Amongst patients with KRAS mutations (72/85), concomitant mutations were observed in TP53 (47/85; 55.3%), CDKN2A (16/85; 18.8%), and SMAD4 (9/85; 10.6%). Herein, we characterized the frequency of somatic mutations associated with DSB repair genes in patients with BRPC/LAPC. Data analysis on outcomes related to radiation response in patients with mutations in DDR pathways is ongoing, but will likely also benefit from multi-institutional efforts to increase the power to answer this question.

Gene expression of non-homologous end-joining pathways in the prognosis of ovarian cancer

Ovarian cancer is the deadliest gynecologic malignancy in women, with a 46% five-year overall survival rate. The objective of the study was to investigate the effects of non-homologous end-joining (NHEJ) genes on clinical outcomes of ovarian cancer patients. To determine if these genes act as prognostic biomarkers of mortality and disease progression, the expression profiles of 48 NHEJ-associated genes were analyzed using an array of statistical and machine learning techniques: logistic regression models, decision trees, naive-Bayes, two sample t-tests, support vector machines, hierarchical clustering, principal component analysis, and neural networks. In this process, the correlation of genes with patient survival and disease progression and recurrence was noted. Also, multiple features from the gene set were found to have significant predictive capabilities. APTX, BRCA1, PAXX, LIG1, and TP53 were identified as most important out of all the candidate genes for predicting clinical outcomes of ovarian cancer patients.

Activation of the DNA damage response pathway in the infected gastric tissue with Helicobacter pylori: a case-control study.

Gastritis is among the most common human diseases worldwide. Although the involvement of Helicobacter pylori infection as a class I human carcinogen for gastric cancer progression is accepted, it is not well known how gastritis progression to atrophy and stomach cancer occurs. In this case-control study, the potential link of H. pylori infection with alteration in the transcription of genes involved in DNA Damage Response pathways was investigated among the patients with gastritis. To measure the difference in the relative mRNA expression level of ATM, CHEK2, TP53, DCLRE1C, POLM, and XRCC4 genes between H. pylori-infected and non-infected patients, gastric biopsies of 30 H. pylori infected patients with moderate chronic gastritis and 30 non-infected patients with mild chronic gastritis were analyzed. Up-regulation of genes linked to non-homologous end joining (NHEJ) pathway (DCLRE1C, POLM, and XRCC) was shown in 40% (8.44 fold ± 13.91), 63.33% (15.72 fold ± 33.08) and 50% (9.99 fold ± 21.55), respectively, and also to DDR pathway (ATM, CHEK2, and TP53) in 33% (2.42 fold ± 3.17), 40% (2.86 fold ± 3.61) and 50% (5.00 fold ± 6.52), respectively. No correlation was detected between alteration in the transcription level of the studied genes and age or gender. Our results provide new data that may support the potential involvement of H. pylori infection in the activation of genes involved in DNA damage response, mainly through a non-homologous end-joining DNA repair system that might be linked to mutagenesis in the pre-cancerous gastric tissue.

Holding it together: DNA end synapsis during non-homologous end joining

DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the majority of these breaks in vertebrates by directly ligating DNA ends back together. Upon formation of a DSB, a multiprotein complex is assembled on DNA ends which tethers them together within a synaptic complex. Synapsis is a critical step of the NHEJ pathway as loss of synapsis can result in mispairing of DNA ends and chromosome translocations. As DNA ends are commonly incompatible for ligation, the NHEJ machinery must also process ends to enable rejoining. This review describes how recent progress in single-molecule approaches and cryo-EM have advanced our molecular understanding of DNA end synapsis during NHEJ and how synapsis is coordinated with end processing to determine the fidelity of repair.

High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition.

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

Rapid Genotyping of Nothobranchius furzeri Embryos, Larvae, and Adult Fish via High-Resolution Melt Analysis (HRMA).

CRISPR-Cas9 has eased the induction of sequence-specific mutations and has therefore become a powerful tool to generate mutant lines for studying the role of specific genes. The cellular repair of Cas9-induced double-stranded DNA breaks by the error-prone nonhomologous end-joining pathway can result in various indel mutations. Having identified and chosen a specific mutation in a target gene, the establishment of a respective mutant line requires a feasible and precise method to differentiate the genotypes of the offspring. Here, we provide a protocol that allows genotyping of large numbers of Nothobranchius furzeri embryos, larvae, and adults harboring a previously identified indel or point mutation in a short time via high-resolution melt analysis (HRMA).

MiR-4796 enhances the sensitivity of breast cancer cells to ionising radiation by impairing the DNA repair pathway.

MicroRNAs (miRNAs) are important regulators of DNA damage response (DDR) through post-transcriptional regulation on their target genes, which are implicated in DDR and DNA repair (DR). In this study, we investigated the functional roles and target genes of miR-4796 and miR-1287 in breast cancer cells in response to radiation. The molecular mechanism of miR-4796 in regulating the radiosensitivity of breast cancer cells was also elucidated. Real-time polymerase chain reaction detected miR-4796 and miR-1287 expression; colony formation assay and irradiation therapy tumour xenograft in vivo examined radiosensitising effect; comet assay assessed DNA damage; immunofluorescence imaging determined the formation of γ-H2AX foci; targetscan and RegRNA predicted target mRNAs; luciferase reporter and mutation assays validated target genes; western blotting detected the expression of genes at the protein level; and flow cytometry quantified the activities of nonhomologous end-joining (NHEJ) and homologous recombination (HR). The expressions of miR-4796 and miR-1287 were acutely fluctuated in response to ionising radiation. In the absence of radiation, overexpression of miR-1287 dramatically promoted growth of breast cancer cells in vitro and in vivo, whereas overexpression of miR-4796 did not affect cell growth. When under the treatment with radiation, overexpression of miR-4796 suppressed DR and sensitised cancer cells to radiation both in vitro and in vivo. However, such effect was only observed in cell assays in the overexpressed miR-1287 group, and not confirmed in vivo. We therefore further explored the molecular mechanism of action of miR-4796, and found that miR-4796 targeted multiple components of DDR and DR, including ATM, BRCA1, PARP and RAD51. Moreover, overexpression of miR-4796 inhibited the expression of these DDR components at the protein level. In addition, miR-4796 inhibited HR and NHEJ repair pathways and aggravated radiation-induced DNA damage. The findings here suggest that miR-4796 can enhance radiation-induced cell death by directly targeting multiple DDR components, and repress NHEJ and HR DNA repair pathways. miR-4796 can act as an effective radiation sensitising agent.

Comparative analysis of the molecular response characteristics in Platycodon grandiflorus irradiated with heavy ion beams and X-rays

The response of plants to radiation is an essential topic in both space plant cultivation and mutation breeding by radiation. In this study, heavy ion beams (HIB) generated by the ground accelerator and X-rays (XR) were used as models of high linear energy transfer (LET) and low LET radiation to study the molecular response mechanism of Platycodon grandiflorus (P. grandiflorus) seedlings after irradiation. The gene and protein expression profiles of P. grandiflorus after 15 Gy HIB and 20 Gy XR radiation were analyzed by transcriptome and proteome. The results showed that the number of differentially expressed genes (DEGs) induced by HIB radiation was less than that of XR group, but HIB radiation induced more differentially expressed proteins (DEPs). Both HIB and XR radiation activated genes of RNA silencing, double-strand break repair and cell catabolic process. DNA replication and cell cycle related genes were down-regulated. The genes of cell wall and external encapsulating structure were up-regulated after HIB radiation. The gene expression of protein folding and glucan biosynthesis increased after XR radiation. Protein enrichment analysis indicated that HIB radiation resulted in differential protein enriched in photosynthesis and secondary metabolite biosynthesis pathways, while XR radiation induced differential protein of glyoxylate and dicarboxylate metabolism and carbon metabolism. After HIB and XR radiation, the genes of antioxidant system and terpenoid and polyketide metabolic pathways presented different expression patterns. HIB radiation led to the enrichment of non-homologous end-joining pathway. The results will contribute to understanding the biological effects of plants under space radiation.