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At least 19 recordsLinked to original sources
PubMed2025

γ-tubulin mediates DNA double-strand break repair.

Double-strand breaks (DSBs) in DNA pose a critical threat to genomic integrity, potentially leading to the onset and progression of various diseases, including cancer. Cellular responses to such lesions entail sophisticated repair mechanisms primarily mediated by non-homologous end joining (NHEJ) and homologous recombination (HR). Interestingly, the efficient recruitment of repair proteins and completion of DSB repair likely involve complex, inter-organelle communication and coordination of cellular components. In this study, we report a role of γ-tubulin in DSB repair. γ-tubulin is a major microtubule nucleation factor governing microtubule dynamics. We show that γ-tubulin is recruited to the site of DNA damage and is required for efficient DSB repair via both NHEJ and HR. Suppression of γ-tubulin impedes DNA repair and exacerbates DNA damage accumulation. Furthermore, γ-tubulin mediates the mobilization and formation of DNA damage foci, which serve as repair centers, thereby facilitating the recruitment of HR and NHEJ repair proteins on damaged chromatin. Finally, pharmacological inhibition of γ-tubulin enhances the cytotoxic effect of DNA-damaging agents, consistent with the DNA repair function of γ-tubulin, and underscoring the potential of its therapeutic intervention in cancer therapy.

DNA Breaks, Double-Stranded↗
PubMed2025

Focus on numbers - characterizing protein accumulation at DNA double-strand breaks.

Unrepaired DNA double-strand breaks can lead to cell death or genomic rearrangements. The DNA damage response (DDR) is a complex signaling cascade in which a plethora of factors act to finely tune repair pathway choice. Several DDR proteins have been shown to accumulate at sites of DNA lesions in characteristic dot-like structures known as DNA repair foci. Changes in foci brightness, commonly expressed in arbitrary intensity units, are often used as readout for DNA repair dynamics. However, due in part to technical challenges, the stoichiometry, absolute number of proteins recruited to DDR foci, and their impact on the resolution of the break remain incompletely characterized. Here, we combine spatial intensity distribution analysis (SpIDA) and a custom foci detection algorithm into an easy-to-use pipeline that, starting from confocal images, allows quantitative description of protein accumulation in DNA repair foci. Moreover, by quantifying foci based on their molecular count, SpIDA overcomes the limitations of ambiguous intensity units, enabling stoichiometric quantification between repair factors and providing a unifying means for experimental comparisons.

DNA Breaks, Double-Stranded↗
PubMed2026

Genetic and physical interactions reveal overlapping and distinct contributions to meiotic double-strand break formation in C. elegans.

Double-strand breaks (DSBs) are the most deleterious lesions experienced by our genome. Yet, DSBs are intentionally induced during gamete formation to promote the exchange of genetic material between homologous chromosomes. While the conserved topoisomerase-like enzyme Spo11 catalyzes DSBs, additional regulatory proteins-referred to as 'Spo11 accessory factors'-regulate the number, timing, and placement of DSBs during meiotic prophase, ensuring that SPO-11 does not wreak havoc on the genome. Despite the importance of the accessory factors, they are poorly conserved at the sequence level, suggesting that these factors may adopt unique functions in different species. In this work, we present a detailed analysis of the genetic and physical interactions between the DSB factors in the nematode Caenorhabditis elegans, providing new insights into conserved and novel functions of these proteins. This work shows that HIM-5 is the determinant of X-chromosome-specific crossovers and that its retention in the nucleus is dependent on DSB-1, the sole accessory factor that interacts with SPO-11. We further provide evidence that HIM-5 mediates interactions with the different accessory factors subgroups, providing insights into how components on the DNA loops may interact with the chromosome axis.

Animals↗
PubMed2026

MRE11 suppresses germline mutagenesis at meiotic double-strand breaks in mice.

SPO11 forms hundreds of double-strand breaks (DSBs) to initiate meiotic recombination that is normally error-free. However, SPO11 activity can be mutagenic when one chromatid incurs closely spaced DSBs (double cuts), especially when DSBs are dysregulated by loss of the ATM kinase. De novo indels and structural variants can arise via end joining at double cuts within a single hotspot (microdeletions) or at adjacent hotspots separated by at least 30 kb, as we now show, sometimes accompanied by ectopic insertions of double-cut fragments. Here, we investigate how meiotic DSB end processing influences end joining. In MRE11-deficient mouse spermatocytes, which do not resect their DSBs, deletions at double cuts occur readily, with end-joining breakpoint profiles closely matching SPO11 DSB profiles. Microdeletions suggest that two DSBs can be as close as ∼21 bp. The tyrosyl-DNA phosphodiesterase TDP2 contributes to both deletion formation and ectopic insertion of double-cut fragments, presumably by removing SPO11 from DNA ends prior to joining. Finally, observations suggest a cooperative role for MRE11 and ATM in locally regulating DSB distributions. Our findings provide insight into the mechanism of de novo mutation origin, emphasizing the role of meiotic DSBs in shaping genome evolution.

Animals↗
PubMed1979

X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution.

This work presents a neutral filter elution method for detecting DNA double strand breaks in mouse L1210 cells after X-ray. The assay will detect the number of double strand breaks induced by as little as 1000 rad of X-ray. The rate of DNA elution through the filters under neutral conditions increases with X-ray dose. Certain conditions for deproteinization, pH, and filter type are shown to increase the assay's sensitivity. Hydrogen peroxide and Bleomycin also induce apparent DNA double strand breaks, although the ratios of double to single strand breaks vary from those produced by X-ray. The introduction of double strand cuts by HpA I restriction endonuclease in DNA lysed on filters results in a rapid rate of elution under neutral conditions, implying that the method can detect double strand breaks if they exist in the DNA. The eluted DNA bands with a double stranded DNA marker in cesium chloride. This evidence suggests that the assay detects DNA double strand breaks. L1210 cells are shown to rejoin most of the DNA double strand breaks induced by 5-10 krad of X-ray with a half-time of about 40 minutes.

Animals↗
PubMed1978

Gamma-ray induced double-strand breaks in DNA resulting from randomly-inflicted single-strand breaks: temporal local denaturation, a new radiation phenomenon?

The induction of single- and double-strand breaks in DNA by gamma-rays has been measured. The maximum number of nucleotide pairs (a) between two independently induced single-strand breaks in opposite strands of the DNA which cannot prevent the occurrence of a double-strand break was found to amount to about 16. This value did not differ significantly for the four types of bacteriophage DNA investigated (T4, T7 and PM2 DNA, and replicative form DNA of phage phiX174) and was the same in 10(-2) M phosphate buffer containing 0, 0.5 or 1 M NaCl. In 10(-3) M phosphate buffer a was 34 nucleotide pairs. Evidence is presented that the relatively large value of a has to be ascribed at least partly to a temporal local denaturation accompanying the induction of a single-strand scission. A contribution of base damage that labilizes the DNA-helix, between two single-strand breaks to the high value of a can not be excluded.

Cobalt Radioisotopes↗
PubMed2025

MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks.

Ubiquitylation is critically implicated in the recognition and repair of DNA double-strand breaks. The adaptor protein MDC1 mediates the recruitment of the key DNA damage responsive E3 ubiquitin ligase RNF8 to the break sites. It does so by directly interacting with RNF8 in a phosphorylation-dependent manner that involves the RNF8 FHA domain, thus initiating targeted chromatin ubiquitylation at the break sites. Here, we report that MDC1 also directly binds to two additional E3 ubiquitin ligases, Pellino 1 and 2, which were recently implicated in the DNA damage response. Through a combination of biochemical, biophysical and X-ray crystallographic approaches, we reveal the molecular details of the MDC1-Pellino complexes. Furthermore, we show that in mammalian cells, MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks by a direct phosphorylation-dependent interaction between the two proteins. Taken together, our findings provide new molecular insights into the ubiquitylation pathways that govern genome stability maintenance.

DNA Breaks, Double-Stranded↗
PubMed1978

The rejoining of DNA double-strand breaks and a model for the formation of chromosomal rearrangements.

The recombination type of process, which has been proposed by Resnick to explain the rejoining of radiation-induced DNA double-strand breaks, is combined with the molecular theory of radiation action to provide a description of the formation of chromosomal rearrangments. It is shown that the majority of chromosomal aberration types found at the first mitosis after radiation can be explained on the basis of one radiation-induced DNA double-strand break in the backbone of the unineme chromatid, followed by the enzymatically controlled recombinational process for the rejoining of the double-strand break. The recombinogenic process for the repair of DNA double-strand breaks relies on the close association between the broken DNA double helix and homologous DNA. The homologous nature of repeated DNA base pair sequences is used, in this model, to explain the occurrence of chromosomal exchanges between non-homologous chromosomes. The important role which repetitive DNA plays in the formation of chromosomal rearrangements and in the distribution of 'break-points' found in radiation experiments is discussed.

Base Sequence↗
PubMed2026

Repair of DNA double-strand breaks after low radiation doses in childhood cancer survivors and matched cancer-free individuals.

DNA double-strand breaks (DSBs) which arise in G1- or G0-phase normal human cells are repaired by nonhomologous end-joining (NHEJ), a pathway which is important for cell survival but can cause mutations at the break sites. DSB repair by NHEJ is very efficient at high damage levels of 1 or more DSBs per cell, much less efficient at lower damage levels and almost absent if only ~0.05 DSBs per cell are induced. Here, we have analyzed the repair of high and low levels of radiation-induced DSBs in primary fibroblasts from 136 childhood cancer survivors, half of whom developed a second independent tumor later in life, and compared it to the response of primary fibroblasts from 68 individually matched cancer-free individuals. We measured the DSB repair efficiency by quantifying residual γH2AX foci with an automated scoring system at 24 h after irradiation with doses of 2.5, 5, 10, and 100 mGy, which induce about 0.0625, 0.125, 0.25, and 2.5 DSBs per cell, respectively. Although childhood cancer survivors and cancer-free individuals repaired DSBs after 10 and 100 mGy equally efficiently, their response to lower doses differed drastically. While repair in cancer-free individuals was inefficient after 2.5 mGy, childhood cancer survivors repaired DSBs after this dose as efficiently as after higher doses. These results indicate that most of the childhood cancer survivors analyzed here may harbor a genetic alteration that affects their response to low levels of DSBs. We suggest that such alterations may be either inherited or caused by previous tumor treatments.

Humans↗
PubMed2025

Arabidopsis thaliana FANCONI ANAEMIA I (FANCI) has roles in the repair of interstrand crosslinks and CRISPR-Cas9 induced DNA double strand breaks.

DNA repair is crucial for genome stability, in particular for plants which are exposed to high levels of damage arising from UV irradiation, soil pollutants and reactive oxygen species. Damage that affects both strands of the DNA duplex is harder to repair due to both the lack of a template strand and the potential for physical separation of fragmented chromosomes. As such, DNA double-strand breaks (DSBs) and interstrand DNA crosslinks (ICL) are particularly cytotoxic forms of damage. Here we report the functions of FANCONI ANAEMIA I (FANCI), an Arabidopsis thaliana homologue of the mammalian ICL repair protein. We show that in plant cells, as in mammals, FANCI forms a nuclear localised complex with FANCD2. Genetic analysis of plants lacking FANCI displays significant hypersensitivity to the DNA crosslinking reagent mitomycin C. Furthermore, mutation of FANCI in combination with mutations in a second ICL repair factor, METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81), results in increased levels of programmed cell death compared to the corresponding single mutants, revealing roles in maintaining plant genome stability. Sequence analysis of mutational repair of CRISPR-Cas9-induced DSBs revealed that FANCI promotes single nucleotide insertions and reduces longer deletions. This pattern of mutations may reflect roles for FA proteins in replication-coupled repair of a subset of DSBs. Taken together, this analysis finds evidence for multiple roles for FANCI in the maintenance of plant genome stability.

Arabidopsis↗
PubMed1977

DNA double-strand breaks and alkali-labile bonds produced by bleomycin.

Both in linear T2 DNA, analyyzed by velocity sedimentation, and in supercoiled Col EL DNA, analyzed by gel electrophoresis, the number of double-strand breaks produced by bleomycin was directly propotional to the number of single-strand breaks and was far greater than the number expected from random coincidence of single-strand breaks, suggesting that the bleomycin-induced double-strand breaks occur as an independent event. In Col EL DNA, at least twice as many single-strand breaks were found under alkaline assay conditions as were found under neutral conditions, showing the production of alkaline-labile bonds by bleomycin.

Bleomycin↗
PubMed2026

Efficient and precise programmable DNA knock-in without double-strand breaks.

Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging1,2. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7 kb to more than 10 kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor 2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-T cell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-T cells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.

Animals↗
PubMed2025

Measuring double-strand break repair events in mammalian cells with multi-target CRISPR.

A mechanistic understanding of the different pathways involved in the repair of DSBs is a timely, yet challenging task. CRISPR-Cas9 is a powerful tool to induce DNA double-strand breaks (DSB) at defined genomic locations to study the ensuing repair response, but Cas9 studies are typically limited by i) low-throughput induction of DSB, by targeting only one or a few genomic sites, or ii) the use of genetically integrated reporter systems, which do not always reflect endogenous phenotypes. To address these limitations, we developed multi-target CRISPR, a Cas9-based tool to controllably induce DSBs in high-throughput at endogenous sites, by leveraging repetitive genomic regions. In this Chapter, we describe how to design and execute a multi-target CRISPR experiment. We also detail how to analyze next-generation sequencing data for characterization of DSB repair events at multiple cut sites. We envision that multi-target CRISPR will become a valuable tool for the study of mammalian DSB repair mechanisms.

DNA Breaks, Double-Stranded↗
PubMed2017

And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break.

Heterochromatin is mostly composed of repeated DNA sequences prone to aberrant recombination. How cells maintain the stability of these sequences during double-strand break (DSB) repair has been a long-standing mystery. Studies in Drosophila cells revealed that faithful homologous recombination repair of heterochromatic DSBs relies on the striking relocalization of repair sites to the nuclear periphery before Rad51 recruitment and repair progression. Here, we summarize our current understanding of this response, including the molecular mechanisms involved, and conserved pathways in mammalian cells. We will highlight important similarities with pathways identified in budding yeast for repair of other types of repeated sequences, including rDNA and short telomeres. We will also discuss the emerging role of chromatin composition and regulation in heterochromatin repair progression. Together, these discoveries challenged previous assumptions that repair sites are substantially static in multicellular eukaryotes, that heterochromatin is largely inert in the presence of DSBs, and that silencing and compaction in this domain are obstacles to repair.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

Animals↗
PubMed2025

Efficiency of DNA repair mechanisms of domestic dog primary fibroblasts isolated from small and large breeds of different ages in response to double stranded breaks (DSB).

Aging is associated with increased genomic instability, a phenomenon largely driven by the accumulation of DNA damage over time, and large species of mammals seem to have more robust DNA repair systems associated with longer lives. Among DNA lesions, double-strand breaks (DSBs) are particularly deleterious and have been implicated in age-related functional decline and disease. In this study, we investigated how age and body mass affect the efficiency of DSB repair (DSBr) in primary fibroblast cells isolated from domestic dogs, a species that exhibits significant intraspecies variation in lifespan and body mass. Primary fibroblast cells were isolated from puppies and senior dogs of both large and small breeds. Cells were treated with 100 µM etoposide to induce DSBs and subsequently analyzed at two post-treatment recovery intervals (2 and 24 h) to correlated with the two pathways associated with DSBr, the fast, non-homologous end joining (NHEJ) and the much slower, homologous recombination (HR). Cells were stained for γ-H2AX foci and images were collected using confocal microscopy. We found that mean fluorescence per cell was higher in older dogs of both size classes in the 2 h recovery, indicating higher amounts of DNA damage but suggesting similar efficiencies through the NHEJ repair mechanism in older dogs despite size class. We also show that mean fluorescence per cell was higher in the older large breed dogs in the 24 h recovery, suggesting that the slower phase associated with HR seems to be deficient in cells from older, larger breeds of dogs. These findings support the broader theory that aging is associated with impaired genomic maintenance and establish domestic dogs as a valuable model for studying the cellular mechanisms of age-related genomic instability.

Animals↗
PubMed1979

DNA damage and repair in relation to cell killing in neocarzinostatin-treated HeLa cells.

To elucidate the mechanism of the cell killing activity of neocarzinostatin on mammalian cells, the drug-induced damage of DNA and its repair were examined. Very low doses of neocarzinostatin, at which high survival of cells was observed, clearly produced single-strand breaks of DNA and decomposition of the 'DNA complex', but these damages appeared to be repaired almost completely. At higher doses of neocarzinostatin, single-strand breaks were repaired to a considerable extent while double-strand breaks seemed not to be repaired. The number of non-repairable single-strand breaks was about twice that of double-strand breaks. This implies that single-strand breaks are repaired except for those constituting double-strand breaks. Although at low levels of neocarzinostatin repair of double-strand breaks may occur, the correlation existing between the colony-forming ability of cells treated with neocarzinostatin and non-repairable DNA breakage suggests that production of a small number of critical non-repairable double-strand breaks per cell may be responsible for the cell killing activity of the drug.

Antibiotics, Antineoplastic↗
PubMed1979

Site specificity of bleomycin-mediated single-strand scissions and alkali-labile damage in duplex DNA.

Form II PM2 DNA, which contained bleomycin-mediated single-strand breaks, was purified and treated with the extracellular endonuclease from Alteromonas BAL 31. This enzyme cleaves the phosphodiester backbone opposite a single-strand break to yield a double-strand break. The locations of these double-strand breaks were determined relative to the cleavage sites produced by the restriction enzyme HindIII. The experimental procedure was as follows. Form I PM2 DNA was treated with bleomycin to produce alkali-labile bonds. These were hydrolyzed by alkali treatment and the DNA, now containing single-strand breaks, was purified and treated with the BAL 31 enzyme and the HindIII enzyme to determine the positions of the original alkali-labile bonds. It was found that the single-strand breaks and alkali-labile bonds were introduced at preferred sites on the PM2 genome, since electrophoretic analyses of the DNA after the HindIII digestion revealed DNA bands of discrete sizes. The molecular weights of the DNA fragments produced by these treatments indicate that single-strand breaks and alkali-labile bonds occur at the same sites as those previously determined for direct double-strand scissions introduced by bleomycin at neutral pH. Some of the specific sites of double-strand scissions mediated by bleomycin at neutral pH (Lloyd et al., 1978b) are also shown here to be relatively more reactive than other sites when the DNA contains superhelical turns.

Bacteriophages↗