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Despite the great advances in discovering cyclic peptides against protein targets, their reduced aqueous solubility, cell permeability, and activity of the cyclic peptide restrict its utilization in advanced biological research and therapeutic applications. Here we report on a novel approach of structural alternation of the exocyclic and linker parts that led to a new derivative with significantly improved cell activity allowing us to dissect its mode of action in detail. We have identified an effective cyclic peptide (CP7) that induces approximately a 9‐fold increase in DNA damage accumulation and a remarkable increase in apoptotic cancer cell death compared to the reported molecule. Notably, treating cells with CP7 leads to a dramatic decrease in the efficiency of non‐homologous end joining (NHEJ) repair of DNA double‐strand breaks (DSBs), which is accompanied by an increase in homologous recombination (HR) repair. Interestingly, treating BRCA1‐deficient cells with CP7 restores HR integrity, which is accompanied by increased resistance to CP7. Additionally, CP7 treatment increases the sensitivity of cancer cells to ionizing radiation. Collectively, our findings demonstrate that CP7 is a selective inhibitor of NHEJ, offering a potential strategy to enhance the effectiveness of radiation therapy. [ABSTRACT FROM AUTHOR]

This article discusses a strategy called the T-circle vector strategy that increases the frequency of site-specific integration (SSI) in soybean plants. The authors used a non-homologous end joining (NHEJ) repair pathway to insert the entire T-DNA into a specific target site in the soybean genome. They tested different vector designs and found that a vector with two target sites and a split selectable marker gene resulted in a higher frequency of SSI events. The T-circle vector strategy has the potential to reduce costs and improve the efficiency of plant production. [Extracted from the article]

Background: Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population‐level trials. As a result, a proportion of patients are under‐ or over‐dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy. Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear. Purpose: The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects. Methods: Normal human cells (RPE1), genetically modified to introduce defects in DNA double‐strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE‐LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12 keV/µm) and alpha particles (129 keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild‐type cells to repair‐deficient cells, and RBE values were calculated as the ratio of the MID of X‐ray and particle irradiated cells. 53BP1 foci were used to quantify radiation‐induced DSBs and their repair following irradiation. Results: Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM−/− SER = 2.0 and Lig4−/− SER = 1.8), with genes associated with HR having smaller effects (BRCA1−/− SER = 1.2). Wild‐type cells showed RBEs of 1.1, 1.3, 5.0 for low‐ and high‐LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR‐defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ‐defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR‐defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects. Conclusions: DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality. [ABSTRACT FROM AUTHOR]

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double‐strand breaks (DSBs) have been implicated in arsenic‐induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As2O3) on four different DSB repair pathways using the human pre‐B cell line Nalm‐6. Random integration analysis showed that As2O3 does not negatively affect non‐homologous end joining or polymerase theta‐mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As2O3 decreases the efficiency of homologous recombination (HR) and, less prominently, single‐strand annealing. Consistent with this finding, As2O3 decreased gene‐targeting efficiency, owing to a significant reduction in the frequency of HR‐mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one‐ended DSBs requiring HR for precise repair. Intriguingly, we found that As2O3 significantly enhances sensitivity to those anticancer agents in HR‐proficient cells. Our results suggest that arsenic‐induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic‐associated carcinogenesis and therapeutic options. [ABSTRACT FROM AUTHOR]

SUMMARY: Non‐homologous end joining (NHEJ) plays a major role in repairing DNA double‐strand breaks and is key to genome stability and editing. The minimal core NHEJ proteins, namely Ku70, Ku80, DNA ligase IV and XRCC4, are conserved, but other factors vary in different eukaryote groups. In plants, the only known NHEJ proteins are the core factors, while the molecular mechanism of plant NHEJ remains unclear. Here, we report a previously unidentified plant ortholog of PAXX, the crystal structure of which showed a similar fold to human 'PAXX'. However, plant PAXX has similar molecular functions to human XLF, by directly interacting with Ku70/80 and XRCC4. This suggests that plant PAXX combines the roles of mammalian PAXX and XLF and that these functions merged into a single protein during evolution. This is consistent with a redundant function of PAXX and XLF in mammals. Significance Statement: Non‐homologous end joining (NHEJ) is a major pathway to repair DNA double‐strand breaks and therefore is important for genome maintenance and editing. Using an evolutional analysis, we identified a plant ortholog of an NHEJ protein PAXX and contributed to understanding the exact molecular mechanism of NHEJ in plants. [ABSTRACT FROM AUTHOR]

Cytosine deaminases AID/APOBEC proteins act as potent nucleic acid editors, playing important roles in innate and adaptive immunity. However, the mutagenic effects of some of these proteins compromise genomic integrity and may promote tumorigenesis. Here, we demonstrate that human APOBEC3G (A3G), in addition to its role in innate immunity, promotes repair of double‐strand breaks (DSBs) in vitro and in vivo. Transgenic mice expressing A3G successfully survived lethal irradiation, whereas wild‐type controls quickly succumbed to radiation syndrome. Mass spectrometric analyses identified the differential upregulation of a plethora of proteins involved in DSB repair pathways in A3G‐expressing cells early following irradiation to facilitate repair. Importantly, we find that A3G not only accelerates DSB repair but also promotes deamination‐dependent error‐free rejoining. These findings have two implications: (a) strategies aimed at inhibiting A3G may improve the efficacy of genotoxic therapies used to cure malignant tumours; and (b) enhancing A3G activity may reduce acute radiation syndrome in individuals exposed to ionizing radiation. [ABSTRACT FROM AUTHOR]

SUMMARY: High efficiency site‐directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR‐associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR‐associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70‐nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site‐directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four‐fold higher than in untethered controls. Integrations via canonical homology‐directed repair or non‐homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN‐associated tethering to improve site‐directed DNA integration in plants. Significance Statement: An exceptionally high rate of site‐directed integration of exogenous DNA into the soybean (Glycine max) genome was achieved using a novel method to increase the local donor DNA concentration around the CRISPR target site of interest. A donor DNA was covalently tethered to a fusion protein between LbCas12a and the HUH endonuclease of Faba Bean Necrotic Yellow Virus, which was delivered into soybean protoplasts and plants as part of a ribonucleoprotein complex. [ABSTRACT FROM AUTHOR]

Non‐homologous end joining (NHEJ) is the main DNA repair mechanism for the repair of double‐strand breaks (DSBs) throughout the course of the cell cycle. DSBs are generated in developing B and T lymphocytes during V(D)J recombination to increase the repertoire of B and T cell receptors. DSBs are also generated during the class switch recombination (CSR) process in mature B lymphocytes, providing distinct effector functions of antibody heavy chain constant regions. Thus, NHEJ is important for both V(D)J recombination and CSR. NHEJ comprises core Ku70 and Ku80 subunits that form the Ku heterodimer, which binds DSBs and promotes the recruitment of accessory factors (e.g., DNA‐PKcs, Artemis, PAXX, MRI) and downstream core factors (XLF, Lig4 and XRCC4). In recent decades, new NHEJ proteins have been reported, increasing complexity of this molecular pathway. Numerous in vivo mouse models have been generated and characterized to identify the interplay of NHEJ factors and their role in development of adaptive immune system. This review summarizes the currently available mouse models lacking one or several NHEJ factors, with a particular focus on early B cell development. We also underline genetic interactions and redundancy in the NHEJ pathway in mice. [ABSTRACT FROM AUTHOR]

FAM35A, alternatively known as SHLD2 and RINN2, was recently characterized as a DNA repair gene, evolutionarily conserved in higher vertebrates. FAM35A is a 53BP1‐pathway factor and a component of the Shieldin/RINN complex. Among 53BP1‐pathway factors, FAM35A has unique domains: an N‐terminal disordered domain and three C‐terminal OB‐fold domains. These C‐terminal domains have homology with the OB‐fold domains of the single‐stranded DNA binding protein, RPA1. With other 53BP1‐pathway factors, FAM35A inhibits DNA end resection. FAM35A defective cell lines are sensitive to DNA double‐strand break inducing agents. Concurrent FAM35A and BRCA1 defects in mammalian cell lines cause resistance to PARP inhibitors and camptothecin. The clinical relevance of this interaction is still unknown, but cancer genomics databases indicate that FAM35A is deleted in 6–13% of prostate cancers and in at least one triple negative breast cancer patient‐derived BRCA1 defective cell line. From meta‐analysis, FAM35A overexpression in patients with triple negative and basal‐like breast cancers is associated with poor survival compared to patients with low expression. From this evidence, clarification of FAM35A's function and the related mechanism of chemoresistance is likely to have clinical implications. [ABSTRACT FROM AUTHOR]

Repressor/activator protein 1 (RAP1) is a highly evolutionarily conserved protein found at telomeres. Although yeast Rap1 is a key telomere capping protein preventing non‐homologous end joining (NHEJ) and consequently telomere fusions, its role at mammalian telomeres in vivo is still controversial. Here, we demonstrate that RAP1 is required to protect telomeres in replicative senescent human cells. Downregulation of RAP1 in these cells, but not in young or dividing pre‐senescent cells, leads to telomere uncapping and fusions. The anti‐fusion effect of RAP1 was further explored in a HeLa cell line where RAP1 expression was depleted through an inducible CRISPR/Cas9 strategy. Depletion of RAP1 in these cells gives rise to telomere fusions only when telomerase is inhibited. We further show that the fusions triggered by RAP1 loss are dependent upon DNA ligase IV. We conclude that human RAP1 is specifically involved in protecting critically short telomeres. This has important implications for the functions of telomeres in senescent cells. Synopsis: Human RAP1 specifically protects critically short telomeres from DNA damage response activation and telomere fusions in replicative senescent fibroblasts and tumor cells upon telomerase inhibition. RAP1 protects critically short telomeres of replicative senescent cells and HeLa cells upon telomerase inhibition.Telomere fusions generated by RAP1 depletion, are dependent on DNA ligase IV.RAP1 downregulation compromises re‐growth of replicative senescent cells upon p21 inhibition. [ABSTRACT FROM AUTHOR]

Structural maintenance of chromosomes flexible hinge domain‐containing protein 1 (SMCHD1) has been implicated in X‐chromosome inactivation, imprinting, and DNA damage repair, and mutations in SMCHD1 can cause facioscapulohumeral muscular dystrophy. More recently, SMCHD1 has also been identified as a component of telomeric chromatin. Here, we report that SMCHD1 is required for DNA damage signaling and non‐homologous end joining (NHEJ) at unprotected telomeres. Co‐depletion of SMCHD1 and the shelterin subunit TRF2 reduced telomeric 3′‐overhang removal in time‐course experiments, as well as the number of chromosome end fusions. SMCHD1‐deficient cells displayed reduced ATM S1981 phosphorylation and diminished formation of γH2AX foci and of 53BP1‐containing telomere dysfunction‐induced foci (TIFs), indicating defects in DNA damage checkpoint signaling. Removal of TPP1 and subsequent activation of ATR signaling rescued telomere fusion events in TRF2‐depleted SMCHD1 knockout cells. Together, these data indicate that SMCHD1 depletion reduces telomere fusions in TRF2‐depleted cells due to defects in ATM‐dependent checkpoint signaling and that SMCHD1 mediates DNA damage response activation upstream of ATM phosphorylation at uncapped telomeres. Synopsis: Loss of the telomere binding protein TRF2 leads to chromosome end‐to‐end fusions mediated by non‐homologous end joining. Here, SMCHD1 (structural maintenance of chromosomes flexible hinge domain‐containing protein 1) is demonstrated to promote the ATM‐dependent DNA damage response at TRF2‐deprived telomeres, which is required for the fusion events. SMCHD1 loss prevents telomere fusions in TRF2‐depleted cells.SMCHD1 is required for the ATM‐dependent DNA damage response in TRF2‐depleted cells.SMCHD1 mediates DNA damage signaling activation upstream of ATM phosphorylation at uncapped telomeres.ATR activation in SMCHD1 knockout cells rescues telomere fusion events in TRF2‐depleted cells. [ABSTRACT FROM AUTHOR]

Synthetic lethality refers to a lethal phenotype that results from the simultaneous disruptions of two genes, while the disruption of either gene alone is viable. Many DNA double strand break repair (DSBR) genes have synthetic lethal relationships with oncogenes and tumor suppressor genes, which can be exploited for targeted cancer therapy, an approach referred to as combination therapy. DNA double-strand breaks (DSBs) are one of the most toxic lesions to a cell and can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR and NHEJ genes are particularly attractive targets for cancer therapy because these genes have altered expression patterns in cancer cells when compared with normal cells and these genetic abnormalities can be targeted for selectively killing cancer cells. Here, we review recent advances in the development of small molecule inhibitors against HR and NHEJ genes to induce synthetic lethality and address the future directions and clinical relevance of this approach. © 2017 IUBMB Life, 69(12):929-937, 2017 [ABSTRACT FROM AUTHOR]

GATAD2B gene is involved in chromatin modification and transcription activity. Loss-of-function mutations of GATAD2B have recently been defined to cause a recognizable syndrome with intellectual disability (ID). Human TPM3 gene encoding thin filament protein is associated with myopathies. Both genes are located on chromosome 1q21.3. We herein report an infant with feeding difficulty, developmental delay, hypotonia, and dysmorphic features including small palpebral fissures, telecanthus, sparse hair and eyebrow, cup-shaped ears, and clinodactyly. Karyotype was normal. Single nucleotide polymorphism array revealed a 1.06 Mb deletion of chromosome 1q21.3, which was confirmed to be de novo. The deleted region encompassed 35 genes, including three known disease-associated genes, namely GATAD2B, TPM3, and HAX1. We further identify and summarize seven additional patients with 1q21.3 microdeletion from literature review and clinical databases (DECIPHER, ISCA/ClinGen). Genomic location analysis of all eight patients revealed different breakpoints and no segmental duplication, indicating that non-homologous end joining is a likely mechanism underlying this particular microdeletion. This data suggests that 1q21.3 microdeletion is a recurrent microdeletion syndrome with distinguishable phenotypes, and loss of function of GATAD2B is the major contributor of the characteristic facies and ID. Additionally, the deletion of TPM3 warrants a risk of concomitant muscle disease in our patient. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Sirtuins, a family of protein deacetylases, promote cellular homeostasis by mediating communication between cells and environment. The enzymatic activity of the mammalian sirtuin SIRT7 targets acetylated lysine in the N-terminal tail of histone H3 (H3K18Ac), thus modulating chromatin structure and transcriptional competency. SIRT7 deletion is associated with reduced lifespan in mice through unknown mechanisms. Here, we show that SirT7-knockout mice suffer from partial embryonic lethality and a progeroid-like phenotype. Consistently, SIRT7-deficient cells display increased replication stress and impaired DNA repair. SIRT7 is recruited in a PARP1-dependent manner to sites of DNA damage, where it modulates H3K18Ac levels. H3K18Ac in turn affects recruitment of the damage response factor 53 BP1 to DNA double-strand breaks ( DSBs), thereby influencing the efficiency of non-homologous end joining ( NHEJ). These results reveal a direct role for SIRT7 in DSB repair and establish a functional link between SIRT7-mediated H3K18 deacetylation and the maintenance of genome integrity. [ABSTRACT FROM AUTHOR]

An X-linked muscular dystrophy, with deficiency of full-length dystrophin and expression of a low molecular weight dystrophin-related protein, has been described in Japanese Spitz dogs. The aim of this study was to identify the causative mutation and develop a specific test to identify affected cases and carrier animals. Gene expression studies in skeletal muscle of an affected animal indicated aberrant expression of the Duchenne muscular dystrophy (dystrophin) gene and an anomaly in intron 19 of the gene. Genome-walking experiments revealed an inversion that interrupts two genes on the X chromosome, the Duchenne muscular dystrophy gene and the retinitis pigmentosa GTPase regulator gene. All clinically affected dogs and obligate carriers that were tested had the mutant chromosome, and it is concluded that the inversion is the causative mutation for X-linked muscular dystrophy in the Japanese Spitz breed. A PCR assay that amplifies mutant and wild-type alleles was developed and proved capable of identifying affected and carrier individuals. Unexpectedly, a 7-year-old male animal, which had not previously come to clinical attention, was shown to possess the mutant allele and to have a relatively mild form of the disease. This observation indicates phenotypic heterogeneity in Japanese Spitz muscular dystrophy, a feature described previously in humans and Golden Retrievers. With the availability of a simple, fast and accurate test for Japanese Spitz muscular dystrophy, detection of carrier animals and selected breeding should help eliminate the mutation from the breed. [ABSTRACT FROM AUTHOR]

Non-homologous end joining ( NHEJ) is the major model proposed for Agrobacterium T- DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA- PKcs, DNA ligase IV ( LIG4), Ataxia telangiectasia mutated ( ATM), and ATM- and Rad3-related ( ATR), play an important role in 'classical' (c) NHEJ. Other proteins, including histone H1 ( HON1), XRCC1, and PARP1, participate in a 'backup' (b) NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant-specific DNA LIGASE VI ( LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild-type plants, suggesting enhanced T- DNA integration in these mutants. These results were confirmed using a promoter-trap transformation vector that requires T- DNA integration into the plant genome to activate a promoterless gusA ( uidA) gene, by virus-induced gene silencing ( VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T- DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T- DNA integration and transgene methylation. A double mutant ( ku80/ parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T- DNA integration. Thus, T- DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration. [ABSTRACT FROM AUTHOR]

The use of TALEN and CRISPR/CAS nucleases is becoming increasingly popular as a means to edit single target sites in one-cell mouse embryos. Nevertheless, an area that has received less attention concerns the engineering of structural genome variants and the necessary religation of two distant double-strand breaks. Herein, we applied pairs of TALEN or sgRNAs and Cas9 to create deletions in the Rab38 gene. We found that the deletion of 3.2 or 9.3 kb, but not of 30 kb, occurs at a frequency of 6–37%. This is sufficient for the direct production of mutants by embryo microinjection. Therefore, deletions up to ∼10 kb can be readily achieved for modeling human disease alleles. This work represents an important step towards the establishment of new protocols that support the ligation of remote DSB ends to achieve even larger rearrangements. [ABSTRACT FROM AUTHOR]

Radiation exposure is a serious threat to biomolecules, particularly DNA, proteins and lipids. Various exogenous substances have been reported to protect these biomolecules. In this study we explored the effect of pre-treatment with G-002M, a mixture of three active derivatives isolated from the rhizomes of Podophyllum hexandrum, on DNA damage response in irradiated human blood leukocytes. Blood was collected from healthy male volunteers, preincubated with G-002M and then irradiated with various doses of radiation. Samples were analyzed using flow cytometry to quantify DNA double strand break (DSB) biomarkers including γ-H2AX, P53BP1 and levels of ligase IV. Blood samples were irradiated in vitro and processed to determine time and dose-dependent kinetics. Semiquantitative RT-PCR was performed at various time points to measure gene expression of DNA -PKcs, Ku80, ATM, and 53BP1; each of these genes is involved in DNA repair signaling. Pre-treatment of blood with G-002M resulted in reduction of γ-H2AX and P53BP1 biomarkers levels and elevated ligase IV levels relative to non-G-002M-treated irradiated cells. These results confirm suppression in radiation-induced DNA DSBs. Samples pre-treated with G-002M and then irradiated also showed significant up-regulation of DNA-PKcs and Ku80 and downregulation of ATM and 53BP1 gene expressions, suggesting that G-002M plays a protective role against DNA damage. The protective effect of G-002M may be due to its ability to scavange radiation-induced free radicals or assist in DNA repair. Further studies are needed to decipher the role of G-002M on signaling molecules involved in radiation-induced DNA damage repair pathways. Environ. Mol. Mutagen. 55:436-448, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3+ transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Transcription activator-like effector (TALE) nucleases (TALENs) have recently emerged as a revolutionary genome editing tool in many different organisms and cell types. The site-specific chromosomal double-strand breaks introduced by TALENs significantly increase the efficiency of genomic modification. The modular nature of the TALE central repeat domains enables researchers to tailor DNA recognition specificity with ease and target essentially any desired DNA sequence. Here, we comprehensively review the development of TALEN technology in terms of scaffold optimization, DNA recognition, and repeat array assembly. In addition, we provide some perspectives on the future development of this technology. Biotechnol. Bioeng. 2013; 110: 1811-1821. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases ( ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3′-phosphotransferase VIII ( aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant ( Pm- R) clones with expressing nucleases. Of these Pm- R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas. [ABSTRACT FROM AUTHOR]

Abstract: The Ku heterodimer, consisting of the proteins Ku70 and Ku80, is the central component of the non-homologous end joining (NHEJ) pathway of double strand break (DSB) repair. Ku is able to recognize and bind a DSB by virtue of its ring-like structure. Both pre-repair and topologically trapped post-repair Ku heterodimers are thought to be inhibitory to multiple cellular processes. Thus, a regulated mechanism for the removal of Ku from chromatin was predicted to exist. Recent evidence shows that Ku80 is removed from DNA through a ubiquitin-mediated process. Similar processes have been shown to be involved in the regulated dissociation of a host of other proteins from chromatin, and this appears to be a general and conserved mechanism for the regulation of chromatin-associated factors. A potential mechanism for this pathway is discussed. [Copyright &y& Elsevier]

Abstract: DNA double-strand breaks resulting from normal cellular processes including replication and exogenous sources such as ionizing radiation pose a serious risk to genome stability, and cells have evolved different mechanisms for their efficient repair. The two major pathways involved in the repair of double-strand breaks in eukaryotic cells are non-homologous end joining and homologous recombination. Numerous factors affect the decision to repair a double-strand break via these pathways, and accumulating evidence suggests these major repair pathways both cooperate and compete with each other at double-strand break sites to facilitate efficient repair and promote genomic integrity. [ABSTRACT FROM AUTHOR]

Abstract: Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25μM etoposide was highly toxic and initiated a double-stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA-PKcs to γH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and γH2AX response to correlate with the inability of the cell to correct topoIIα-initiated DNA damage. γH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre-treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIα-dependent DNA defects within topologically open chromatin domains. [Copyright &y& Elsevier]

Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of ∼15% of X- or γ-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ. [ABSTRACT FROM AUTHOR]

The induction of double-strand breaks (DSBs) in plant genomes can lead to increased homologous recombination or site-specific mutagenesis at the repair site. This phenomenon has the potential for use in gene targeting applications in plant cells upon the induction of site-specific genomic DSBs using zinc finger nucleases (ZFNs). Zinc finger nucleases are artificial restriction enzymes, custom-designed to cleave a specific DNA sequence. The tools and methods for ZFN assembly and validation could potentially boost their application for plant gene targeting. Here we report on the design of biochemical and in planta methods for the analysis of newly designed ZFNs. Cloning begins with de novo assembly of the DNA-binding regions of new ZFNs from overlapping oligonucleotides containing modified helices responsible for DNA-triplet recognition, and the fusion of the DNA-binding domain with a FokI endonuclease domain in a dedicated plant expression cassette. Following the transfer of fully assembled ZFNs into Escherichia coli expression vectors, bacterial lysates were found to be most suitable for in vitro digestion analysis of palindromic target sequences. A set of three in planta activity assays was also developed to confirm the nucleic acid digestion activity of ZFNs in plant cells. The assays are based on the reconstruction of GUS expression following transient or stable delivery of a mutated uidA and ZFN-expressing cassettes into target plants cells. Our tools and assays offer cloning flexibility and simple assembly of tested ZFNs and their corresponding target sites into Agrobacterium tumefaciens binary plasmids, allowing efficient implementation of ZFN-validation assays in planta. [ABSTRACT FROM AUTHOR]

The ability of plants to repair DNA double-strand breaks (DSBs) is essential for growth and fertility. The Arabidopsis DSB repair proteins AtRAD50 and AtMRE11 form part of an evolutionarily conserved complex that, in Saccharomyces cerevisiae and mammals, includes a third component termed XRS2 and NBS1, respectively. The MRN complex (MRX in yeast) has a direct role in DSB repair and is also required for DNA damage signaling and checkpoint activation in a pathway mediated by the protein kinase ATM. This study characterizes Arabidopsis and maize NBS1 orthologues that share conserved protein motifs with human NBS1. Both plant NBS1 proteins interact with the corresponding MRE11 orthologues, and deletion analysis of AtNBS1 defines a region towards the C-terminus (amino acids 465–500) that is required for interaction with AtMRE11. Arabidopsis lines homozygous for a T-DNA insertional mutation in AtNBS1 display hypersensitivity to the DNA cross-linking reagent mitomycin C, and this phenotype can be rescued by complementation with the wild-type gene, consistent with a function for AtNBS1 in plant DSB repair. Analysis of atnbs1-1 atatm double mutants revealed a role for AtNBS1 in meiotic recombination. While atatm mutants produce reduced seed numbers, plants deficient in both AtATM and AtNBS1 are completely infertile. Cytological analysis of these double mutants revealed incomplete chromosome pairing and synapsis in meiotic prophase, and extensive chromosome fragmentation in metaphase I and subsequent stages. These results suggest a novel role for AtNBS1 that is independent of AtATM-mediated signaling and functions in the very early stages of meiosis. [ABSTRACT FROM AUTHOR]

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases. [ABSTRACT FROM AUTHOR]

t(11;18)(q21;q21) is the most frequent chromosomal aberration specifically associated with mucosa-associated lymphoid tissue (MALT) lymphoma. The translocation fuses the API2 gene to the MALT1 gene and generates a functional API2-MALT1 transcript. The breakpoint of the fusion gene is well characterized at the transcript level but poorly understood at the genomic level and the mechanism underlying the translocation is unknown. We identified the genomic breakpoint in 19 t(11;18)-positive MALT lymphoma cases by polymerase chain reaction and sequencing and analysed the junctional sequences. The breakpoints were scattered in intron 7 and exon  8 of the API2 gene, and introns  4, 6, 7 and 8 of the MALT1 gene. Comparative sequence analysis between the API2-MALT1 fusion on der(11) and the MALT1-API2 fusion on der(18) showed extensive alterations including deletions, duplications and non-template-based insertions at the fusion junctions in all cases examined. An extensive sequence search failed to reveal any known sequence motifs that might be associated with chromosomal recombination or any novel consensus sequences at or near the breakpoints on both der(11) and der(18) except in one case, in which Alu repeats spanned the breakpoint of the MALT1-API2 fusion. Our results suggest that t(11;18) may result from illegitimate non-homologous end joining following double strand breaks. [ABSTRACT FROM AUTHOR]

We have examined the genetic requirements for efficient repair of a site-specific DNA double-strand break (DSB) in Schizosaccharomyces pombe. Technology was developed in which a unique DSB could be generated in a non-essential minichromosome, Ch16, using the Saccharomyces cerevisiae HO-endonuclease and its target site, MA Ta. DSB repair in this context was predominantly through interchromosomal gene conversion. We found that the homologous recombination (HR) genes rhp51+, rad22A+, rad32+ and the nucleotide excision repair gene rad16+ were required for efficient interchromosomal gene conversion. Further, DSB-induced cell cycle delay and efficient HR required the DNA integrity checkpoint gene rad3+. Rhp55 was required for interchromosomal gene conversion; however, an alternative DSB repair mechanism was used in an rhp55EΔ background involving ku70+ and rhp51+. Surprisingly, DSB-induced minichromosome toss was significantly reduced in ku7OΔ and lig4Δ non-homologous end joining (NHEJ) mutant backgrounds compared with wild type. Furthermore, roles for Ku70 and Lig4 were identified in suppressing DSB-induced chromosomal rearrangements associated with gene conversion. These findings are consistent with both competitive and cooperative interactions between components of the HR and NHEJ pathways. [ABSTRACT FROM AUTHOR]

Summary Double-strand breaks (DSBs) in DNA may occur spontaneously in the cell or be induced experimentally by γ-irradiation, and represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination appears to be the major pathway for DSB repair in humans and plants, and it may also be the major route whereby T-DNA integrates into the plant genome during cell transformation. In yeast and mammals, the exposed ends of damaged DNA are bound with high affinity by a dimer of Ku70 and Ku80 proteins, which protects the ends from exonucleases and juxtaposes the two ends of the DSB, independent of sequence homology. Here we report the functional characterization of Ku70 and Ku80 from Arabidopsis thaliana , and demonstrate that AtKu80 and AtKu70 form a heterodimer with DNA binding activity that is specific for DNA ends. An atku80 knockout mutant shows hypersensitivity to the DNA-damaging agents menadione and bleomycin, consistent with a role for AtKu80 in the repair of DSBs in vivo in Arabidopsis . [ABSTRACT FROM AUTHOR]

Genome editing with engineered nucleases (GEENs) introduce site‐specific DNA double‐strand breaks (DSBs) and repairs DSBs via nonhomologous end‐joining (NHEJ) pathways that eventually create indels (insertions/deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENs to DNA may interfere with binding of key components of DNA repair complexes, favoring either classical‐ or alternative‐NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w [ABSTRACT FROM AUTHOR]

DNA double‐strand breaks are a threat to genome integrity and cell viability. The nucleolytic processing of broken DNA ends plays a central role in dictating the repair processes that will mend these lesions. Usually, DNA end resection promotes repair by homologous recombination, whereas minimally processed ends are repaired by non‐homologous end joining. Important in this process is the chromatin‐binding protein 53BP1, which inhibits DNA end resection. How 53BP1 shields DNA ends from nucleases has been an enduring mystery. The recent discovery of shieldin, a four‐subunit protein complex with single‐stranded DNA‐binding activity, illuminated a strong candidate for the ultimate effector of 53BP1‐dependent end protection. Shieldin consists of REV7, a known 53BP1‐pathway component, and three hitherto uncharacterized proteins: C20orf196 (SHLD1), FAM35A (SHLD2), and CTC‐534A2.2 (SHLD3). Shieldin promotes many 53BP1‐associated activities, such as the protection of DNA ends, non‐homologous end joining, and immunoglobulin class switching. This review summarizes the identification of shieldin and the various models of shieldin action and highlights some outstanding questions requiring answers to gain a full molecular understanding of shieldin function. [ABSTRACT FROM AUTHOR]