Multiple DNA Double-strand Breaks Research Articles

Heavy water inhibits DNA double-strand break repairs and disturbs cellular transcription, presumably via quantum-level mechanisms of kinetic isotope effects on hydrolytic enzyme reactions.

Heavy water, containing the heavy hydrogen isotope, is toxic to cells, although the underlying mechanism remains incompletely understood. In addition, certain enzymatic proton transfer reactions exhibit kinetic isotope effects attributed to hydrogen isotopes and their temperature dependencies, indicative of quantum tunneling phenomena. However, the correlation between the biological effects of heavy water and the kinetic isotope effects mediated by hydrogen isotopes remains elusive. In this study, we elucidated the kinetic isotope effects arising from hydrogen isotopes of water and their temperature dependencies in vitro, focusing on deacetylation, DNA cleavage, and protein cleavage, which are crucial enzymatic reactions mediated by hydrolysis. Intriguingly, the intracellular isotope effects of heavy water, related to the in vitro kinetic isotope effects, significantly impeded multiple DNA double-strand break repair mechanisms crucial for cell survival. Additionally, heavy water exposure enhanced histone acetylation and associated transcriptional activation in cells, consistent with the in vitro kinetic isotope effects observed in histone deacetylation reactions. Moreover, as observed for the in vitro kinetic isotope effects, the cytotoxic effect on cell proliferation induced by heavy water exhibited temperature-dependency. These findings reveal the substantial impact of heavy water-induced isotope effects on cellular functions governed by hydrolytic enzymatic reactions, potentially mediated by quantum-level mechanisms underlying kinetic isotope effects.

Inducible Expression ofthe Restriction Enzyme Uncovered Genome-Wide Distribution andDynamic Behavior ofHistones H4K16ac andH2A.Z at DNA Double-Strand Breaks inArabidopsis.

DNA double-strand breaks (DSBs) are among the most serious types of DNA damage, causing mutations and chromosomal rearrangements. In eukaryotes, DSBs are immediately repaired in coordination with chromatin remodeling for the deposition of DSB-related histone modifications and variants. To elucidate the details of DSB-dependent chromatin remodeling throughout the genome, artificial DSBs need to be reproducibly induced at various genomic loci. Recently, a comprehensive method for elucidating chromatin remodeling at multiple DSB loci via chemically induced expression of a restriction enzyme was developed in mammals. However, this DSB induction system is unsuitable for investigating chromatin remodeling during and after DSB repair, and such an approach has not been performed in plants. Here, we established a transgenic Arabidopsis plant harboring a restriction enzyme gene Sbf I driven by a heat-inducible promoter. Using this transgenic line, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of histones H4K16ac and H2A.Z and investigated the dynamics of these histone marks around the endogenous 623 Sbf I recognition sites. We also precisely quantified DSB efficiency at all cleavage sites using the DNA resequencing data obtained by the ChIP-seq procedure. From the results, Sbf I-induced DSBs were detected at 360 loci, which induced the transient deposition of H4K16ac and H2A.Z around these regions. Interestingly, we also observed the co-localization of H4K16ac and H2A.Z at some DSB loci. Overall, DSB-dependent chromatin remodeling was found to be highly conserved between plants and animals. These findings provide new insights into chromatin remodeling that occurs in response to DSBs in Arabidopsis.

225Ac]Ac- and [111In]In-DOTA-trastuzumab theranostic pair: cellular dosimetry and cytotoxicity in vitro and tumour and normal tissue uptake in vivo in NRG mice with HER2-positive human breast cancer xenografts

BackgroundTrastuzumab (Herceptin) has improved the outcome for patients with HER2-positive breast cancer (BC) but brain metastases (BM) remain a challenge due to poor uptake of trastuzumab into the brain. Radioimmunotherapy (RIT) with trastuzumab labeled with α-particle emitting, 225Ac may overcome this challenge by increasing the cytotoxic potency on HER2-positive BC cells. Our first aim was to synthesize and characterize [111In]In-DOTA-trastuzumab and [225Ac]Ac-DOTA-trastuzumab as a theranostic pair for imaging and RIT of HER2-positive BC, respectively. A second aim was to estimate the cellular dosimetry of [225Ac]Ac-DOTA-trastuzumab and determine its cytotoxicity in vitro on HER2-positive BC cells. A third aim was to study the tumour and normal tissue uptake of [225Ac]Ac-DOTA-trastuzumab using [111In]In-DOTA-trastuzumab as a radiotracer in vivo in NRG mice with s.c. 164/8-1B/H2N.luc+ human BC tumours that metastasize to the brain.ResultsTrastuzumab was conjugated to 12.7 ± 1.2 DOTA chelators and labeled with 111In or 225Ac. [111In]In-DOTA-trastuzumab exhibited high affinity specific binding to HER2-positive SK-BR-3 human BC cells (KD = 1.2 ± 0.3 × 10–8 mol/L). Treatment with [225Ac]Ac-DOTA-trastuzumab decreased the surviving fraction (SF) of SK-BR-3 cells dependent on the specific activity (SA) with SF < 0.001 at SA = 0.74 kBq/µg. No surviving colonies were noted at SA = 1.10 kBq/µg or 1.665 kBq/µg. Multiple DNA double-strand breaks (DSBs) were detected in SK-BR-3 cells exposed to [225Ac]Ac-DOTA-trastuzumab by γ-H2AX immunofluorescence microscopy. The time-integrated activity of [111In]In-DOTA-trastuzumab in SK-BR-3 cells was measured and used to estimate the absorbed doses from [225Ac]Ac-DOTA-trastuzumab by Monte Carlo N-Particle simulation for correlation with the SF. The dose required to decrease the SF of SK-BR-3 cells to 0.10 (D10) was 1.10 Gy. Based on the D10 reported for γ-irradiation of SK-BR-3 cells, we estimate that the relative biological effectiveness of the α-particles emitted by 225Ac is 4.4. Biodistribution studies in NRG mice with s.c. 164/8-1B/H2N.luc+ human BC tumours at 48 h post-coinjection of [111In]In-DOTA-trastuzumab and [225Ac]Ac-DOTA-trastuzumab revealed HER2-specific tumour uptake (10.6 ± 0.6% ID/g) but spleen uptake was high (28.9 ± 7.4% ID/g). Tumours were well-visualized by SPECT/CT imaging using [111In]In-DOTA-trastuzumab.ConclusionWe conclude that [225Ac]Ac-DOTA-trastuzumab exhibited potent and HER2-specific cytotoxicity on SK-BR-3 cells in vitro and HER2-specific uptake in s.c. 164/8-1B/H2N.luc+ human BC tumours in NRG mice, and these tumours were imaged by SPECT/CT with [111In]In-DOTA-trastuzumab. These results are promising for combining [111In]In-DOTA-trastuzumab and [225Ac]Ac-DOTA-trastuzumab as a theranostic pair for imaging and RIT of HER2-positive BC.

Sequence and chromatin features guide DNA double-strand break resection initiation

DNA double-strand breaks (DSBs) are cytotoxic genome lesions that must be accurately and efficiently repaired to ensure genome integrity. In yeast, the Mre11-Rad50-Xrs2 (MRX) complex nicks 5'-terminated DSB ends to initiate nucleolytic processing of DSBs for repair by homologous recombination. How MRX-DNA interactions support 5' strand-specific nicking and how nicking is influenced by the chromatin context have remained elusive. Using a deep sequencing-based assay, we mapped MRX nicks at single-nucleotide resolution next to multiple DSBs in the yeast genome. We observed that the DNA end-binding Ku70-Ku80 complex directed DSB-proximal nicks and that repetitive MRX cleavage extended the length of resection tracts. We identified a sequence motif and a DNA meltability profile that is preferentially nicked by MRX. Furthermore, we found that nucleosomes as well as transcription impeded MRX incisions. Our findings suggest that local DNA sequence and chromatin features shape the activity of this central DSB repair complex.

Carbon ion radiation and clustered DNA double-strand breaks.

A carbon ion categorized as a heavy ion particle has been used for cancer radiotherapy. High linear energy transfer (LET) carbon ion irradiation deposits energy at a high density along a particle track, generating multiple types of DNA damage. Complex DNA lesions, comprising DNA double-strand breaks (DSBs), single-strand breaks, and base damage within 1-2 helical turns (<3-4nm), are thought to be difficult to repair and critically influence cell viability. In addition to the effect of lesion complexity, the most recent studies have demonstrated another characteristic of high LET particle radiation-induced DNA damage, clustered DSBs. Clustered DSBs are defined as the formation of multiple DSBs in close proximity where the scale of clustering is approximately 1-2μm3, i.e., the scale of the event is estimated to be > ∼1Mbp. This chapter reviews the hallmarks of clustered DSBs and how such DNA damage influences genome instability and cell viability in the context of high LET carbon ion radiotherapy.

Monitoring Nuclease Activity by X-Ray Scattering Interferometry Using Gold Nanoparticle-Conjugated DNA.

The biologically critical, exquisite specificity and efficiency of nucleases, such as those acting in DNA repair and replication, often emerge in the context of multiple other macromolecules. The evolved complexity also makes biologically relevant nuclease assays challenging and low-throughput. Meiotic recombination 11 homolog 1 (MRE11) is an exemplary nuclease that initiates DNA double-strand break (DSB) repair and processes stalled DNA replication forks. Thus, DNA resection by MRE11 nuclease activity is critical for multiple DSB repair pathways as well as in replication. Traditionally, in vitro nuclease activity of purified enzymes is studied either through gel-based assays or fluorescence-based assays like fluorescence resonance energy transfer (FRET). However, adapting these methods for a high-throughput application such as inhibitor screening can be challenging. Gel-based approaches are slow, and FRET assays can suffer from interference and distance limitations. Here we describe an alternative methodology to monitor nuclease activity by measuring the small-angle X-ray scattering (SAXS) interference pattern from gold nanoparticles (Au NPs) conjugated to 5'-ends of dsDNA using X-ray scattering interferometry (XSI). In addition to reporting on the enzyme activity, XSI can provide insight into DNA-protein interactions, aiding in the development of inhibitors that trap enzymes on the DNA substrate. Enabled by efficient access to synchrotron beamlines, sample preparation, and the feasibility of high-throughput XSI data collection and processing pipelines, this method allows for far greater speeds with less sample consumption than conventional SAXS techniques. The reported metrics and methods can be generalized to monitor not only other nucleases but also most other DNA-protein interactions.

Engineered sex ratio distortion by X-shredding in the global agricultural pest Ceratitis capitata

BackgroundGenetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits.ResultsHere, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~ 80%).ConclusionWe provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.

Single-Strand Annealing in Cancer.

DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.

RB Regulates DNA Double Strand Break Repair Pathway Choice by Mediating CtIP Dependent End Resection.

Inactivation of the retinoblastoma tumor suppressor gene (RB1) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.

LncRNA SLC26A4-AS1 suppresses the MRN complex-mediated DNA repair signaling and thyroid cancer metastasis by destabilizing DDX5.

Lymph node metastasis is the major adverse feature for recurrence and death of thyroid cancer patients. To identify lncRNAs involved in thyroid cancer metastasis, we systemically screened differentially expressed lncRNAs in lymph node metastasis, thyroid cancer, and normal tissues via RNAseq. We found that lncRNA SLC26A4-AS1 was continuously, significantly down-regulated in normal tissues, thyroid cancer, and lymph node metastasis specimens. Low SLC26A4-AS1 levels in tissues were significantly associated with poor prognosis of thyroid cancer patients. LncRNA SLC26A4-AS1 markedly inhibited migration, invasion, and metastasis capability of cancer cells in vitro and in vivo. Intriguingly, SLC26A4-AS1 could simultaneously interact with DDX5 and the E3 ligase TRIM25, which promoting DDX5 degradation through the ubiquitin-proteasome pathway. In particular, SLC26A4-AS1 inhibited expression of multiple DNA double-strand breaks (DSBs) repair genes, especially genes coding proteins in the MRE11/RAS50/NBS1 (MRN) complex. Enhanced interaction between DDX5 and transcriptional factor E2F1 due to silencing of SLC26A4-AS1 promoted binding of the DDX5-E2F1 complex at promoters of the MRN genes and, thus, stimulate the MRN/ATM dependent DSB signaling and thyroid cancer metastasis. Our study uncovered new insights into the biology driving thyroid cancer metastasis and highlights potentials of lncRNAs as future therapeutic targets again cancer metastasis.

Proline-rich protein PRR19 functions with cyclin-like CNTD1 to promote meiotic crossing over in mouse

Orderly chromosome segregation is enabled by crossovers between homologous chromosomes in the first meiotic division. Crossovers arise from recombination-mediated repair of programmed DNA double-strand breaks (DSBs). Multiple DSBs initiate recombination, and most are repaired without crossover formation, although one or more generate crossovers on each chromosome. Although the underlying mechanisms are ill-defined, the differentiation and maturation of crossover-specific recombination intermediates requires the cyclin-like CNTD1. Here, we identify PRR19 as a partner of CNTD1. We find that, like CNTD1, PRR19 is required for timely DSB repair and the formation of crossover-specific recombination complexes. PRR19 and CNTD1 co-localise at crossover sites, physically interact, and are interdependent for accumulation, indicating a PRR19-CNTD1 partnership in crossing over. Further, we show that CNTD1 interacts with a cyclin-dependent kinase, CDK2, which also accumulates in crossover-specific recombination complexes. Thus, the PRR19-CNTD1 complex may enable crossover differentiation by regulating CDK2.

Centromere scission drives chromosome shuffling and reproductive isolation

A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promoting speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans The resulting DSBs were repaired in a complex manner, leading to the formation of multiple interchromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced ∼3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.

Highly Efficient Multiplexed Base Editing with Minimized Off-Targets for the Development of Universal CAR-T Cells to Treat Pediatric T-ALL

Autologous CAR-T therapies have demonstrated remarkable efficacy in treating some hematologic cancers. While these therapies can have substantial clinical benefit for patients, generating bespoke cell therapies creates manufacturing challenges, resulting in inconsistent products and delays in treatment that are often incompatible with effective clinical management of patients. Strategies to create universally-compatible CAR-T therapies, generated from single donors for the treatment of many patients, have been developed as a solution to these challenges, thereby reducing cost of goods, lot-to-lot variability and enabling timely treatment. Mitigating the risks of graft-versus-host-disease (GvHD) and host rejection of CAR-Ts are important components of any strategy to generate these universal therapies. Most first generation approaches utilize DNA double strand break (DSB)-inducing nucleases to ablate the expression of relevant genes in donor T cells to overcome these barriers. However, simultaneous induction of multiple DSBs results in a cell population containing genomic rearrangements, and can lead to significantly reduced cell proliferation. Approaches to develop CAR-T therapies for T cell malignancies, such as T-ALL, encounter additional challenges, including extensive fratricide caused by targeting T cell surface markers such as CD3 and CD7, which are present on both the malignant and the CAR-T cells. Resolving this issue requires additional gene editing, leading to, in the case of nuclease-based strategies, an increased number of DSBs, further genomic rearrangements, and decreased cell expansion. Base editors (BEs) are a novel class of gene editing reagents that enable programmable, single-base changes in genomic DNA without creating DSBs. Work in the Qasim lab has demonstrated proof of concept for an alternative means of producing universal, fratricide-resistant CAR-T cells for treating T cell malignancies by using cytosine base editor (CBE) technology. Here, we demonstrate a previously-unpublished CBE that produces greatly diminished off-target effects while preserving on-target activity. Using multiplex base editing, we demonstrate simultaneous knockout of four genes (TRAC, CD7, CD52, and PDCD1) with between 80-95% efficiency, producing engineered CAR-T cells with greatly diminished risk of GvHD, graft cell rejection, fratricide, and exhaustion. We show that, in contrast to nuclease editing, concurrent modification of four genetic loci using our reduced off-target CBE produces highly efficient gene knockouts with no detectable genomic rearrangements and no observable change in cell expansion compared to control conditions. T-ALL is a heterogeneous disease with variable expression of CD3 and CD7 across tumor cells in the same patient. To reduce the risk of antigen escape by tumor cells during the course of treatment with base edited CAR-Ts, we envisioned creating two independent CAR-T populations targeting CD3 (3CAR-Ts) or CD7 (7CAR-Ts). Alone or in combination, base edited 3CAR-Ts and 7CAR-Ts demonstrate robust cytokine release, potent in vitro cytotoxicity, and in vivo tumor control with antigen-positive tumor cells, and display minimal antigen-independent activity. Taken together, our approach addresses existing limitations in CAR-T cell manufacturing and demonstrates that simultaneous base editing using an improved specificity CBE at four target genes is a feasible strategy for generating universal, fratricide-resistant CAR-T cells for the potential treatment of T cell malignancies such as T-ALL. More generally, this program demonstrates the potential for base editing to create highly-engineered cell therapies featuring at least four simultaneous edits, which can confer a wide range of desirable therapeutic attributes. Disclosures Qasim: CellMedica: Research Funding; Bellicum: Research Funding; Servier: Research Funding; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Autolus: Equity Ownership.

Dose, LET and Strain Dependence of Radiation-Induced 53BP1 Foci in 15 Mouse Strains Ex Vivo Introducing Novel DNA Damage Metrics.

We present a comprehensive comparative analysis on the repair of radiation-induced DNA damage ex vivo in 15 strains of mice, including 5 inbred reference strains and 10 collaborative-cross strains, of both sexes, totaling 5 million skin fibroblast cells imaged by three-dimensional highthroughput conventional microscopy. Non-immortalized primary skin fibroblasts derived from 76 mice were subjected to increasing doses of both low- and high-LET radiation (X rays; 350 MeV/n 40Ar; 600 MeV/n 56Fe), which are relevant to carcinogenesis and human space exploration. Automated image quantification of 53BP1 radiation-induced foci (RIF) formation and repair during the first 4-48 h postirradiation was performed as a function of dose and LET. Since multiple DNA double-strand breaks (DSBs) are induced in a dose- and LET-dependent manner, our data suggest that when DSBs are formed within the same discrete nuclear region, referred to as the "repair domain", novel mathematical formalisms used to report RIF allowed us to conclude that multiple DSBs can be present in single RIF. Specifically, we observed that the number of RIF per Gy was lower for higher X-ray doses or higher LET particles (i.e., 600 MeV/n 56Fe), suggesting there are more DSBs per RIF when the local absorbed dose increases in the nucleus. The data also clearly show that with more DSBs per RIF, it becomes more difficult for cells to fully resolve RIF. All 15 strains showed the same dose and LET dependence, but strain differences were preserved under various experimental conditions, indicating that the number and sizes of repair domains are modulated by the genetic background of each strain.

Acceleration of cancer science with genome editing and related technologies.

Genome editing includes various edits of the genome, such as short insertions and deletions, substitutions, and chromosomal rearrangements including inversions, duplications, and translocations. These variations are based on single or multiple DNA double‐strand break (DSB)‐triggered in cellulo repair machineries. In addition to these “conventional” genome editing strategies, tools enabling customized, site‐specific recognition of particular nucleic acid sequences have been coming into wider use; for example, single base editing without DSB introduction, epigenome editing with recruitment of epigenetic modifiers, transcriptome engineering using RNA editing systems, and in vitro detection of specific DNA and RNA sequences. In this review, we provide a quick overview of the current state of genome editing and related technologies that multilaterally contribute to cancer science.

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

Processing-Challenges Generated by Clusters of DNA Double-Strand Breaks Underpin Increased Effectiveness of High-LET Radiation and Chromothripsis.

Whereas most endogenous and exogenous DNA damaging agents typically generate lesions that are relatively isolated and can be repaired easily, ionizing radiation (IR) also induces clustered lesions causing DNA double strand breaks (DSBs). Moreover, forms of IR characterized by high linearenergytransfer (LET) induce not only isolated DSBs but also DSB clusters- multiple DSBs in close proximity -that pose increased risks for the cell. DSB clusters can destabilize chromatin locally and compromise processing of individual DSBs within the cluster. Since the discovery of chromothripsis, a phenomenon whereby multiple DSBs locally generated by a catastrophic event causes genomic rearrangements that feed carcinogenesis, DSB clusters receive increased attention also in the field of cancer. While formation of DSB clusters after exposure to high LET is a direct and inherent consequence of the spatial distribution of the constituting energy deposition events, also called track structure, the sources of local genomic shattering underpinning chromothripsis are under investigation. Notably, many consequences of DSB clusters in the affected genome reflect processing by pathways that have evolved to repair DSBs, but which operate with widely different degrees of fidelity. The molecular underpinnings and the basis of the underlying repair pathway choices that ultimately lead to the observed consequences from DSB clusters remain unknown. We developed a tractable model of DSB clustering that allows direct analysis in cells of the consequences of certain configurations of DSB clusters. We outline the rationale for the development of this model and describe its key characteristics. We summarize results suggesting that DSB clusters compromise the first-line DSB-processing pathways of c-NHEJ and HRR, increasing as a consequence the contribution of alt-EJ, which has high propensity of generating chromosomal rearrangements. The results suggest a mechanism for the increased toxicity of high LET radiation and the extensive genomic rearrangements associated with chromothripsis.

3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation.

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1–2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

Induction of somatic mutations by low-dose X-rays: the challenge in recognizing radiation-induced events.

It is difficult to distinguish radiation-induced events from spontaneous events during induction of stochastic effects, especially in the case of low-dose or low-dose-rate exposures. By using a hypersensitive system for detecting somatic mutations at the HPRT1 locus, we investigated the frequency and spectrum of mutations induced by low-dose X-rays. The mutant frequencies induced by doses of >0.15 Gy were statistically significant when compared with the spontaneous frequency, and a clear dose dependency was also observed for mutant frequencies at doses of >0.15 Gy. In contrast, mutant frequencies at doses of <0.1 Gy occurred at non-significant levels. The mutation spectrum in HPRT-deficient mutants revealed that the type of mutations induced by low-dose exposures was similar to that seen in spontaneous mutants. An apparent change in mutation type was observed for mutants induced by doses of >0.2 Gy. Our observations suggest that there could be a critical dose for mutation induction at between 0.1 Gy and 0.2 Gy, where mutagenic events are induced by multiple DNA double-strand breaks (DSBs). These observations also suggest that low-dose radiation delivered at doses of <0.1 Gy may not result in DSB-induced mutations but may enhance spontaneous mutagenesis events.

Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.