Absence Of DNA Double-strand Breaks Research Articles

Assembly of Baculovirus Vectors for Multiplexed Prime Editing.

Efficient genome editing by using CRISPR technologies requires simultaneous and efficient delivery of multiple genetically encoded components to mammalian cells. Amongst all editing approaches, prime editing (PE) has the unique potential to perform seamless genome rewriting, in the absence of DNA double-strand breaks (DSBs). The cargo capacity required for efficient PE delivery to mammalian cells stands at odd with the limited packaging capacity of traditional viral delivery vectors. By contrast, baculovirus (BV) has a large synthetic DNA capacity and can efficiently transduce mammalian cells. Here we describe a protocol for the assembly of baculovirus vectors for multiplexed prime editing in mammalian cells.

Functional restoration of a CFTR splicing mutation through RNA delivery of CRISPR adenine base editor

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is a quite frequent defect causing an aberrant splicing and a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to correct the mutation in the absence of DNA double-strand breaks (DSB). To select the strategy, we developed a minigene cellular model reproducing the 2789+5G>A splicing defect. We obtained up to 70% editing in the minigene model by adapting the ABE to the PAM sequence optimal fortargeting 2789+5G>A with a SpCas9-NG (NG-ABE). Nonetheless, the on-target base correction was accompanied by secondary (bystander) A-to-G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To decrease the bystander edits, we used a specific ABE (NG-ABEmax), which was delivered as mRNA. The NG-ABEmax RNA approach was validated in patient-derived rectal organoids and bronchial epithelial cells showing sufficient gene correction to recover the CFTR function. Finally, in-depth sequencing revealed high editing precision genome-wide and allele-specific correction. Here we report the development of a base editing strategy to precisely repair the 2789+5G>A mutation resulting in restoration of the CFTR function, while reducing bystander and off-target activities.

The Technologist's Dilemma

The Technologist's Dilemma

Differentiated function and localisation of SPO11-1 and PRD3 on the chromosome axis during meiotic DSB formation in Arabidopsis thaliana.

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.

H2A.X Phosphorylation in Oxidative Stress and Risk Assessment in Plasma Medicine.

At serine139-phosphorylated gamma histone H2A.X (γH2A.X) has been established over the decades as sensitive evidence of radiation-induced DNA damage, especially DNA double-strand breaks (DSBs) in radiation biology. Therefore, γH2A.X has been considered a suitable marker for biomedical applications and a general indicator of direct DNA damage with other therapeutic agents, such as cold physical plasma. Medical plasma technology generates a partially ionized gas releasing a plethora of reactive oxygen and nitrogen species (ROS) simultaneously that have been used for therapeutic purposes such as wound healing and cancer treatment. The quantification of γH2A.X as a surrogate parameter of direct DNA damage has often been used to assess genotoxicity in plasma-treated cells, whereas no sustainable mutagenic potential of the medical plasma treatment could be identified despite H2A.X phosphorylation. However, phosphorylated H2A.X occurs during apoptosis, which is associated with exposure to cold plasma and ROS. This review summarizes the current understanding of γH2A.X induction and function in oxidative stress in general and plasma medicine in particular. Due to the progress towards understanding the mechanisms of H2A.X phosphorylation in the absence of DSB and ROS, observations of γH2A.X in medical fields should be carefully interpreted.

Absence of DNA double-strand breaks in human peripheral blood mononuclear cells after 3 Tesla magnetic resonance imaging assessed by γH2AX flow cytometry

Magnetic resonance imaging (MRI) is regarded as a non-harming and non-invasive imaging modality with high tissue contrast and almost no side effects. Compared to other cross-sectional imaging modalities, MRI does not use ionising radiation. Recently, however, strong magnetic fields as applied in clinical MRI scanners have been suspected to induce DNA double-strand breaks in human lymphocytes. In this study we investigated the impact of 3-T cardiac MRI examinations on the induction of DNA double-strand breaks in peripheral mononuclear cells by γH2AX staining and flow cytometry analysis. The study cohort consisted of 73 healthy non-smoking volunteers with 36 volunteers undergoing CMRI and 37 controls without intervention. Differences between the two cohorts were analysed by a mixed linear model with repeated measures. Both cohorts showed a significant increase in the γH2AX signal from baseline to post-procedure of 6.7 % (SD 7.18 %) and 7.8 % (SD 6.61 %), respectively. However, the difference between the two groups was not significant. Based on our study, γH2AX flow cytometry shows no evidence that 3-T MRI examinations as used in cardiac scans impair DNA integrity in peripheral mononuclear cells. • No evidence for DNA double-strand breaks after cardiac MRI. • Prospective study underlines safe use of MRI with regard to DNA damage. • Controlled trial involving both genders investigating DNA DSBs after 3-T MRI.

Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development.

Error-prone repair of DNA double-strand breaks (DSBs) has been postulated to occur through classical non-homologous end joining (NHEJ) in systems ranging from nematode somatic tissues to zebrafish embryos. Contrary to this model, we show that zebrafish embryos mutant for DNA polymerase theta (Polq), a critical component of alternative end joining (alt-EJ), cannot repair DSBs induced by CRISPR/Cas9 or ionizing radiation. In the absence of DSBs, polq mutants are phenotypically normal, but they do not survive mutagenesis and display dramatic differences in the mutation profiles compared with the wild-type. These results show that alt-EJ repair is essential and dominant during the early development of a vertebrate.

Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development

Error-prone repair of DNA double-strand breaks (DSBs) has been postulated to occur through classical non-homologous end joining (NHEJ) in systems ranging from nematode somatic tissues to zebrafish embryos. Contrary to this model, we show that zebrafish embryos mutant for DNA polymerase theta (Polq), a critical component of alternative end joining (alt-EJ), cannot repair DSBs induced by CRISPR/Cas9 or ionizing radiation. In the absence of DSBs, polq mutants are phenotypically normal, but they do not survive mutagenesis and display dramatic differences in the mutation profiles compared with the wild-type. These results show that alt-EJ repair is essential and dominant during the early development of a vertebrate.

The key role of CYC2 during meiosis in Tetrahymena thermophila.

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5–3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0254-9) contains supplementary material, which is available to authorized users.

Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination

The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

Distinct histone modifications define initiation and repair of meiotic recombination in the mouse

Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11(-/-) mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.

Radiation induction of delayed recombination in Schizosaccharomyces pombe

Ionizing radiation is known to induce delayed chromosome and gene mutations in the descendants of the irradiated tissue culture cells. Molecular mechanisms of such delayed mutations are yet to be elucidated, since high genomic complexity of mammalian cells makes it difficult to analyze. We now tested radiation induction of delayed recombination in the fission yeast Schizosaccharomyces pombe by monitoring the frequency of homologous recombination after X-irradiation. A reporter with 200bp tandem repeats went through spontaneous recombination at a frequency of 1.0×10−4, and the frequency increased dose-dependently to around 10×10−4 at 500Gy of X-irradiation. Although the repair of initial DNA damage was thought to be completed before the restart of cell division cycle, the elevation of the recombination frequency persisted for 8–10 cell generations after irradiation (delayed recombination). The delayed recombination suggests that descendants of the irradiated cells keep a memory of the initial DNA damage which upregulates recombination machinery for 8–10 generations even in the absence of DNA double-strand breaks (DSBs). Since radical scavengers were ineffective in inhibiting the delayed recombination, a memory by continuous production of DNA damaging agents such as reactive oxygen species (ROS) was excluded. Recombination was induced in trans in a reporter on chromosome III by a DNA DSB at a site on chromosome I, suggesting the untargeted nature of delayed recombination. Interestingly, Rad22 foci persisted in the X-irradiated population in parallel with the elevation of the recombination frequency. These results suggest that the epigenetic damage memory induced by DNA DSB upregulates untargeted and delayed recombination in S. pombe.

What histone code for DNA repair?

Chromatin structure plays a key role in most processes involving DNA metabolism. Chromatin modifications implicated in transcriptional regulation are relatively well characterized and are thought to be the result of a code on the histone proteins (histone code). This code, involving phosphorylation, ubiquitylation, sumoylation, acetylation and methylation, is believed to regulate chromatin accessibility either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin. Recent evidences suggest that such mechanisms are also involved in DNA damage detection and DNA repair. One of the most well-characterized modifications is caused by the formation of DNA double strand breaks (DSBs), resulting in phosphorylation of histone H2AX (the so-called gamma-H2AX) on the chromatin surrounding the DNA lesion. It is generally believed that histone H2AX phosphorylation is required for the concentration and stabilization of DNA repair proteins to the damaged chromatin. The phosphorylation of this histone seems to play a role in both non-homologous end-joining (NHEJ) and homologous recombination (HR) repair pathways. However, the choice of the repair pathway might depend on or induce additional post-translational modifications affecting other histone proteins necessary to the completion of the entire DNA repair process. Interestingly, even in the absence of DSBs, histone modifications occur. Indeed, following UV-exposure, histone acetylation takes place and is believed to facilitate the nucleotide excision repair (NER) process by promoting chromatin accessibility to the repair factors. This review focuses on recent data characterizing the function of histone modification in various repair processes and discusses if the combination of such modifications can be the trademark of a specific DNA repair pathway.

Ciprofloxacin-induced G2 arrest and apoptosis in TK6 lymphoblastoid cells is not dependent on DNA double-strand break formation

Drugs developed for the treatment of conditions other than neoplasia can also show promise as potential antitumor agents. The fluoroquinolone antibiotic ciprofloxacin (CPFX) is known to modulate cycle cell progression and apoptosis in cancer cells, and is thought to induce DNA double-strand breaks (DSBs) via topoisomerase II (topo II) inhibition and stabilized cleavage complex (SCC) formation. DSBs trigger Ser-139 phosphorylation of histone H2AX (γH2AX) by PI-3-like kinases including ATM; γH2AX can serve as a marker of DNA damage when measured in situ using immunocytochemistry and flow cytometry. The aim of the present study was to investigate the relationship between CPFX-mediated DNA damage and induction of apoptosis in human lymphoblastoid cells and phytohaemagglutinin (PHA)-stimulated lymphocytes (Lymphs). Treatment of TK6 cells (wild-type p53) with 100 μg/ml CPFX for 2-10 h produced no increase in γH2AX; to the contrary, its level in S phase cells was reduced at 10 h compared to controls. Nevertheless, stabilization of topo IIα, ATM Ser-1981 phosphorylation and G2 arrest was observed in TK6 cells exposed to CPFX for ≥4 h. However, following 24 h treatment, γH2AX was dramatically increased in a sub-population of cells indicating the onset of apoptosis (confirmed by presence of activated caspase 3). CPFX had a similar lack of effect on induction of γH2AX at early time points in WTK1 and NH32 cells (devoid of functional p53) and proliferating Lymphs, however, induction of apoptosis was less pronounced than in TK6 cells. Formation of SCC and activation of ATM (but lack of γH2AX induction) indicates topo II-mediated chromatin or DNA changes in the absence of DSBs; ATM activation apparently triggers the G2M checkpoint leading to G2 arrest. The subsequent induction of apoptosis appears to be facilitated by functional p53. CPFX may therefore have a potential use as a chemotherapeutic agent in the treatment of lymphoblast-derived cancer.

Tid1/Rdh54 promotes dissociation of Dmc1 from nonrecombinogenic sites on meiotic chromatin

The meiosis-specific recombinase Dmc1 plays a critical role in DNA strand exchange in budding yeast. Tid1/Rdh54, a member of the Swi2/Snf2 family of DNA translocases, has been shown to stimulate Dmc1-dependent recombination. Tid1and its budding yeast paralog Rad54 have a variety of biochemical activities that may contribute to their biological function. Here we demonstrate that Dmc1 can associate with chromatin in the absence of DNA double-strand breaks (DSBs), and Tid1 suppresses this association. Chromatin immunoprecipitation experiments indicate that an activity shared by Tid1 and Rad54 is required for normal assembly of Dmc1 at DSB sites in preparation for recombination. These results lead to a model in which the ATP hydrolysis-dependent DNA translocase activity of Tid1 acts to promote dissociation of Dmc1 from nonreombinogenic sites on chromatin, with Rad54 being able to substitute for this function in the absence of Tid1. The tendency of Dmc1 to form unproductive interactions with chromatin is proposed to be a consequence of the mechanism of strand exchange. The results raise the possibility that ATP hydrolysis-dependent disruption of nonproductive recombinase-DNA interactions is a feature shared with other homologous recombination systems.