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Charged-particle radiotherapy (CPRT) utilizing low and high linear energy transfer (low-/high-LET) ionizing radiation (IR) is a promising cancer treatment modality having unique physical energy deposition properties. CPRT enables focused delivery of a desired dose to the tumor, thus achieving a better tumor control and reduced normal tissue toxicity. It increases the overall radiation tolerance and the chances of survival for the patient. Further improvements in CPRT are expected from a better understanding of the mechanisms governing the biological effects of IR and their dependence on LET. There is increasing evidence that high-LET IR induces more complex and even clustered DNA double-strand breaks (DSBs) that are extremely consequential to cellular homeostasis, and which represent a considerable threat to genomic integrity. However, from the perspective of cancer management, the same DSB characteristics underpin the expected therapeutic benefit and are central to the rationale guiding current efforts for increased implementation of heavy ions (HI) in radiotherapy. Here, we review the specific cellular DNA damage responses (DDR) elicited by high-LET IR and compare them to those of low-LET IR. We emphasize differences in the forms of DSBs induced and their impact on DDR. Moreover, we analyze how the distinct initial forms of DSBs modulate the interplay between DSB repair pathways through the activation of DNA end resection. We postulate that at complex DSBs and DSB clusters, increased DNA end resection orchestrates an increased engagement of resection-dependent repair pathways. Furthermore, we summarize evidence that after exposure to high-LET IR, error-prone processes outcompete high fidelity homologous recombination (HR) through mechanisms that remain to be elucidated. Finally, we review the high-LET dependence of specific DDR-related post-translational modifications and the induction of apoptosis in cancer cells. We believe that in-depth characterization of the biological effects that are specific to high-LET IR will help to establish predictive and prognostic signatures for use in future individualized therapeutic strategies, and will enhance the prospects for the development of effective countermeasures for improved radiation protection during space travel.

ABSTRACT Agrobacterium‐mediated T‐DNA integration into plant genomes represents a cornerstone for transgenic expression in plant basic research and synthetic biology. However, random T‐DNA integration can disrupt essential endogenous genes or compromise transgene expression, stressing the need for targeted integration strategies. Here we explored CRISPR‐aided targeted T‐DNA integration (CRISTTIN) in Arabidopsis, leveraging CRISPR‐induced double‐strand breaks (DSBs) to facilitate precise T‐DNA insertion. Contrary to our initial hypothesis, conventional Cas9 outperformed a designed Cas9‐adaptor fusion nuclease that may recruit Agrobacterium VirD2/T‐DNA complexes to DSB sites via the adaptor‐VirD2 interaction. Using Cas9‐based CRISTTIN, we streamlined the parallel generation of FERONIA null alleles and in‐locus complementation alleles expressing a wild‐type or mutated gene. This enabled phenotypic comparisons under identical genomic contexts and significantly accelerated gene characterisation and critical residue identification. Additionally, CRISTTIN was employed to simultaneously knockout AGAMOUS and in‐locus integrate a RUBY reporter, yielding plants with pink double‐petaled flowers. CRISTTIN also enabled site‐specific insertion of 35S enhancers for transcriptional upregulation of adjacent genes or reporter constructs for promoter activity monitoring. CRISTTIN's effectiveness was further validated in rice. These results demonstrated CRISTTIN as a versatile tool for gene functional studies and precise control of transgene expression in plants.

DNA double‐strand breaks (DSBs) seriously damage DNA and promote genomic instability that can lead to cell death. They are the source of conditions such as carcinogenesis and aging, but also have important applications in cancer therapy. Therefore, rapid detection and quantification of DSBs in cells are necessary for identifying carcinogenic and anticancer factors. In this study, we detected DSBs using a flow cytometry‐based high‐throughput method to analyze γH2AX intensity. We screened a chemical library containing 9600 compounds and detected multiple DNA‐damaging compounds, although we could not identify mechanisms of action through this procedure. Thus, we also profiled a representative compound with the highest DSB potential, DNA‐damaging agent‐1 (DDA‐1), using a bioinformatics‐based method we termed “molecular profiling.” Prediction and verification analysis revealed DDA‐1 as a potential inhibitor of topoisomerase IIα, different from known inhibitors such as etoposide and doxorubicin. Additional investigation of DDA‐1 analogs and xenograft models suggested that DDA‐1 is a potential anticancer drug. In conclusion, our findings established that combining high‐throughput DSB detection and molecular profiling to undertake phenotypic analysis is a viable method for efficient identification of novel DNA‐damaging compounds for clinical applications. Phenotypic screening for DNA double‐strand breaks detected multiple DNA‐damaging compounds from a library of 9600 compounds. Molecular profiling identified a potential inhibitor of topoisomerase II alpha as an anticancer drug.

Sperm DNA injury is one of the common causes of male infertility. Folic acid deficiency would increase the methylation level of the important genes, including those involved in DNA double‐strand break (DSB) repair pathway. In the early stages, we analysed the correlation between seminal plasma folic acid concentration and semen parameters in 157 infertility patients and 91 sperm donor volunteers, and found that there was a significant negative correlation between seminal folic acid concentration and sperm DNA Fragmentation Index (DFI; r = −0.495, p < 0.01). Then through reduced representation bisulphite sequencing, global DNA methylation of sperm of patients in the low folic acid group and the high folic acid group was analysed, it was found that the methylation level in Rad54 promoter region increased in the folic acid deficiency group compared with the normal folic acid group. Meanwhile, the results of animal model and spermatocyte line (GC‐2) also found that folic acid deficiency can increase the methylation level in Rad54 promoter region, increased sperm DFI in mice, increased the expression of γ‐H2AX, that is, DNA injury marker protein, and increased sensitivity of GC‐2 to external damage and stimulation. The study indicates that the expression of Rad54 is downregulated by folic acid deficiency via DNA methylation. This may be one of the mechanisms of sperm DNA damage caused by folate deficiency.

To overcome genotoxicity, cells have evolved powerful and effective mechanisms to detect and respond to DNA lesions. RecQ Like Helicase‐4 (RECQL4) plays a vital role in DNA damage responses. RECQL4 is recruited to DNA double‐strand break (DSB) sites in a poly(ADP‐ribosyl)ation (PARylation)‐dependent manner, but the mechanism and significance of this process remain unclear. Here, we showed that the domain of RECQL4 recruited to DSBs in a PARylation‐dependent manner directly interacts with poly(ADP‐ribose) (PAR) and contains a PAR‐binding motif (PBM). By replacing this PBM with a PBM of hnRNPA2 or its mutated form, we demonstrated that the PBM in RECQL4 is required for PARylation‐dependent recruitment and the roles of RECQL4 in the DSB response. These results suggest that the direct interaction of RECQL4 with PAR is critical for proper cellular response to DSBs and provide insights to understand PARylation‐dependent control of the DSB response and cancer therapeutics using PARylation inhibitors. Here, we showed that RECQL4 protein contains a poly(ADP‐ribose)‐binding motif (PBM). The interaction of RECQL4 with poly(ADP‐ribose) via its PBM is critical for recruitment to DNA double‐strand break (DSB) sites and proper cellular response to DSBs. These results provide insights for understanding the poly(ADP‐ribosyl)ation‐dependent control of the DSB response and cancer therapeutics using poly(ADP‐ribosyl)ation inhibitors.

Abstract In all living organisms, the response to double-strand breaks (DSBs) is critical for the maintenance of chromosome integrity. Homologous recombination (HR), which utilizes a homologous template to prime DNA synthesis and to restore genetic information lost at the DNA break site, is a complex multistep response. In Bacillus subtilis, this response can be subdivided into five general acts: (1) recognition of the break site(s) and formation of a repair center (RC), which enables cells to commit to HR; (2) end-processing of the broken end(s) by different avenues to generate a 3′-tailed duplex and RecN-mediated DSB ‘coordination’; (3) loading of RecA onto single-strand DNA at the RecN-induced RC and concomitant DNA strand exchange; (4) branch migration and resolution, or dissolution, of the recombination intermediates, and replication restart, followed by (5) disassembly of the recombination apparatus formed at the dynamic RC and segregation of sister chromosomes. When HR is impaired or an intact homologous template is not available, error-prone nonhomologous end-joining directly rejoins the two broken ends by ligation. In this review, we examine the functions that are known to contribute to DNA DSB repair in B. subtilis, and compare their properties with those of other bacterial phyla. Double strand break repair in bacteria

The human RAD52 protein, which forms an oligomeric ring structure, is involved in DNA double‐strand break repair. The N‐terminal half of RAD52 is primarily responsible for self‐oligomerisation and DNA binding. Crystallographic studies have revealed the detailed structure of the N‐terminal half. However, only low‐resolution structures have been reported for the full‐length protein, and thus the structural role of the C‐terminal half in self‐oligomerisation has remained elusive. In this study, we determined the solution structure of the human RAD52 protein by cryo‐electron microscopy (cryo‐EM), at an average resolution of 3.5 Å. The structure revealed an undecameric ring that is nearly identical to the crystal structures of the N‐terminal half. The cryo‐EM map for the C‐terminal half was poorly defined, indicating that the region is intrinsically disordered. The present cryo‐EM structure provides important insights into the mechanistic roles played by the N‐terminal and C‐terminal halves of RAD52 during DNA double‐strand break repair. The cryo‐EM structure of the human DNA repair protein RAD52 was determined at near‐atomic resolution, which revealed that the full‐length protein oligomerises into an undecameric ring. The N‐terminal half is responsible for the oligomerisation. By contrast, the C‐terminal half was not visible in the structure, which is consistent with the predicted intrinsic disorder of the region.

Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts to the phosphorylated histones H2AX (γH2AX) repair foci which are multiproteic nuclear complexes responsible for DSB sensing and signalling. However, neither the components of these foci nor their role are yet known in plants. In this paper, we describe the effects of γH2AX deficiency in Arabidopsis thaliana plants challenged with DSBs in terms of genotoxic sensitivity and E2F-mediated transcriptional responses. We further establish the existence, restrictive to the G1/S transition, of specific DSB-induced foci containing tobacco E2F transcription factors, in both A. thaliana roots and BY-2 tobacco cells. These E2F foci partially colocalize with γH2AX foci while their formation is ataxia telangiectasia mutated (ATM)-dependent, requires the E2F transactivation domain with its retinoblastoma-binding site and is optimal in the presence of functional H2AXs. Overall, our results unveil a new interplay between plant H2AX and E2F transcriptional activators during the DSB response.

DNA double‐strand breaks (DSBs) are highly cytotoxic lesions, and unrepaired or misrepaired DSBs can lead to various human diseases, including immunodeficiency, neurological abnormalities, growth retardation, and cancer. Nonhomologous end joining (NHEJ) is the major DSB repair pathway in mammals. Ku70 and Ku80 are DSB sensors that facilitate the recruitment of downstream factors, including protein kinase DNA‐dependent protein kinase, catalytic subunit (DNA‐PKcs), structural components [X‐ray repair cross‐complementing protein 4 (XRCC4), XRCC4‐like factor (XLF), and paralogue of XRCC4 and XLF (PAXX)], and DNA ligase IV (LIG4), which complete DNA repair. DSBs also trigger the activation of the DNA damage response pathway, in which protein kinase ataxia‐telangiectasia mutated (ATM) phosphorylates multiple substrates, including histone H2AX. Traditionally, research on NHEJ factors was performed using in vivo mouse models and murine cells. However, the current knowledge of the genetic interactions between NHEJ factors in human cells is incomplete. Here, we obtained genetically modified human HAP1 cell lines, which lacked one or two NHEJ factors, including LIG4, XRCC4, XLF, PAXX, DNA‐PKcs, DNA‐PKcs/XRCC4, and DNA‐PKcs/PAXX. We examined the genomic instability of HAP1 cells, as well as their sensitivity to DSB‐inducing agents. In addition, we determined the genetic interaction between XRCC4 paralogues (XRCC4, XLF, and PAXX) and DNA‐PKcs. We found that in human cells, XLF, but not PAXX or XRCC4, genetically interacts with DNA‐PKcs. Moreover, ATM possesses overlapping functions with DNA‐PKcs, XLF, and XRCC4, but not with PAXX in response to DSBs. Finally, NHEJ‐deficient HAP1 cells show increased chromosomal and chromatid breaks, when compared to the WT parental control. Overall, we found that HAP1 is a suitable model to study the genetic interactions in human cells. Nonhomologous end joining is the major DNA double‐strand break repair pathway that includes several factors with complex genetic interactions. X‐ray repair cross‐complementing protein 4 (XRCC4) paralogues include paralogue of XRCC4 and XRCC4‐like factor (XLF; PAXX), XLF, and XRCC4. In human HAP1 cell lines, the severity order of mutant phenotypes is PAXX∆ < XLF∆ < XRCC4∆. XLF has redundant functions with both DNA‐dependent protein kinase, catalytic subunit and ataxia‐telangiectasia mutated (ATM) kinases, while XRCC4 functions redundantly with ATM.

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. Cells have developed a variety of mechanisms for repairing DNA molecules when breaks occur in one or both of the DNA strands. However, we know relatively little about the causes of these breaks, which often occur naturally, or even about how common they are. Learning more about the most common forms of DNA breakage is important because the genomic changes caused by these breaks are driving forces behind both cancer and evolution, including the evolution of drug resistance in bacteria. Shee et al. have developed a new method for detecting double-strand breaks in both bacterial and mammalian cells. The method involved combining a natural virus protein called Gam with a fluorescent protein called GFP (short for green fluorescent protein) to make a fusion protein called GamGFP. Gam was chosen because it binds only to double-strand breaks, traps double-strand breaks, and does not bind to any proteins. Genetic engineering techniques were used to introduce GamGFP into cells, with DNA breaks in these cells showing up as fluorescent spots when viewed under a microscope. Shee et al. used this approach to detect double-strand breaks in both Escherichia coli cells and mammalian cells, and to measure the rate of spontaneous DNA breakage in E. coli. The number of double-strand breaks in E. coli was proportional to the number of times the cells had divided, which provides support for DNA replication-dependent models of spontaneous DNA breakage. The GamGFP method also provided various insights into DNA breaks in mouse and human cells. In particular, Shee et al. found evidence for a mechanism of DNA breakage that appears to be specific to primates. This mechanism involves an enzyme that is only found in the innate immune system of primates removing an amine group from a cytosine. In future, this approach might allow the trapping, mapping and quantification of DNA breaks in all kinds of cells, and the highly specific way GamGFP binds to breaks could make it the preferred tool for studying DNA breakage in mammalian cells.