Search

CRISPR-Cas systems revolutionized the genome engineering field but need to induce double-strand breaks (DSBs) and may be difficult to deliver due to their large protein size. Tn7-like transposons such as CRISPR-associated transposons (CASTs) can be repurposed for RNA-guided DSB-free integration, and obligate mobile element guided activity (OMEGA) proteins of the IS200/IS605 transposon family have been developed as hypercompact RNA-guided genome editing tools. CASTs and OMEGA are exciting, innovative genome engineering tools that can improve the precision and efficiency of editing. This review explores the recent developments and uses of CASTs and OMEGA in genome editing across prokaryotic and eukaryotic cells. The pros and cons of these transposon-based systems are deliberated in comparison to other CRISPR systems.

Although self-incompatibility in apples (Malus × domestica Borkh.) is regulated by a single S-locus with multiple S-haplotypes that comprise pistil S (S-RNase) and pollen S genes, it is not desirable in commercial orchards because it requires cross-pollination to achieve stable fruit production. Therefore, it is important to identify and characterize self-compatible apple cultivars. However, little is known about self-compatibility (SC) and its underlying molecular mechanisms in apples. In this study, we discovered that ‘Vered’, an early maturing and low chilling-requiring apple cultivar, exhibits stable SC, which was evaluated via self-pollination tests. The S-genotype of ‘Vered’ was designated as S24S39sm. Results of genetic analysis of selfed progeny of ‘Vered’ revealed that SC is associated with the S39sm-haplotype, and molecular analyses indicated that it is caused by alternative splicing and a 205-bp deletion in S39sm-RNase. These events induce frameshifts and ultimately produce the defective S39sm-RNase isoforms that lack their C-terminal half. These results enabled us to develop a 117-bp DNA marker that can be used to assist in the selection of self-compatible apples with the dysfunctional S39sm-RNase. Thus, analysis of ‘Vered’ provided insights into the molecular mechanism of the very rare trait of natural stylar-part SC. Moreover, ‘Vered’ is a valuable genetic resource for breeding cultivars with SC and/or low chilling requirement in apple. Our findings contribute to a better understanding of self-compatible molecular mechanisms in apple and provide for the accelerated breeding of self-compatible apple cultivars.Key message: This study identifies the apple cultivar, ‘Vered’, as a stylar-part self-compatible genetic resource. We reveal the molecular mechanism underlying self-compatibility and develop an efficient selection marker for the self-compatibility. [ABSTRACT FROM AUTHOR]

ABSTRACT Background DNA double‐strand breaks are known to be the most lethal kind of DNA damage as they cause genomic instability. Visualization and quantification of these breaks are possible via staining of the phosphorylated histone H2A variant H2AX at serine 139 with the anti‐phospho‐histone H2AX (γ‐H2AX) antibody. Methods The literature was searched with the following keywords: DNA damage, double‐strand break, PBMC, DNA repair, H2AX, γ‐H2AX. Discussion This review discusses various methods for the quantification of γ‐H2AX and the use of γ‐H2AX in cell culture, tissue biopsies, and peripheral blood mononuclear cells. The current research into basal DNA damage as quantified by γ‐H2AX is discussed in relation to cancer. Conclusions This review suggests that γ‐H2AX has the potential for use in the prediction of cancer risk when applied to healthy tissue and peripheral blood mononuclear cells.

Eukaryotic cells encounter diverse threats jeopardizing their integrity, prompting the development of defense mechanisms against these stressors. Among these mechanisms, inflammasomes are well‐known for their roles in coordinating the inflammatory response against infections. Extensive research has unveiled their multifaceted involvement in cellular processes beyond inflammation. Recent studies emphasize the intricate relationship between the inflammasome and the DNA damage response (DDR). They highlight how the DDR participates in inflammasome activation and the reciprocal impact of inflammasome on DDR and genome integrity preservation. Moreover, novel functions of inflammasome sensors in DDR pathways have emerged, broadening our understanding of their roles. Finally, this review delves into identifying common signals that drive the activation of inflammasome sensors alongside activation cues for the DNA damage response, offering potential insights into shared regulatory pathways between these critical cellular processes. [ABSTRACT FROM AUTHOR]

Small fractions of patients suffer from radiotherapy late severe adverse events (AEs Grade ≥ 3), which are usually irreversible and badly affect their quality of life. A novel functional DNA repair assay characterizing several steps of double-strand break (DSB) repair mechanisms was used. DNA repair activities of peripheral blood mononuclear cells were monitored for 1 week using NEXT-SPOT assay in 177 breast and prostate cancer patients. Only seven patients had Grade ≥ 3 AEs, 6 months after radiotherapy initiation. The machine learning method established the importance of variables among demographic, clinical and DNA repair data. The most relevant ones, all related to DNA repair, were employed to build a predictor. Predictors constructed with random forest and minimum bounding sphere predicted late Grade ≥ 3 AEs with a sensitivity of 100% and specificity of 77.17 and 86.22%, respectively. This multiplex functional approach strongly supports a dominant role for DSB repair in the development of chronic AEs. It also showed that affected patients share specific features related to functional aspects of DSB repair. This strategy may be suitable for routine clinical analysis and paves the way for modelling DSB repair associated with severe AEs induced by radiotherapy. [ABSTRACT FROM AUTHOR]

Nonobstructive azoospermia (NOA), the most severe manifestation of male infertility, lacks a comprehensive understanding of its genetic etiology. Here, a bi‐allelic loss‐of‐function variant in REC114 (c.568C > T: p.Gln190*) were identified through whole exome sequencing (WES) in a Chinese NOA patient. Testicular histopathological analysis and meiotic chromosomal spread analysis were conducted to assess the stage of spermatogenesis arrested. Co‐immunoprecipitation (Co‐IP) and Western blot (WB) were used to investigate the influence of variant in vitro. In addition, our results revealed that the variant resulted in truncated REC114 protein and impaired interaction with MEI4, which was essential for meiotic DNA double‐strand break (DSB) formation. As far as we know, this study presents the first report that identifies REC114 as the causative gene for male infertility. Furthermore, our study demonstrated indispensability of the REC114‐MEI4 complex in maintaining DSB homoeostasis, and highlighted that the disruption of the complex due to the REC114 variant may underline the mechanism of NOA. [ABSTRACT FROM AUTHOR]

Radiation therapy aims to maximize doses to cancer cells while minimizing damage to normal tissues. Today, nanoparticles containing high-atomic-number elements, such as gold, gadolinium, and silver, have proven effective as radiosensitizers in radiotherapy to enhance dose delivery for cancer treatment. In this study, we used the Geant4-DNA toolkit to investigate the effects of multiple nanoparticles (NPs) with varying sizes (radius= 3.15 to 5 nm) on DNA damage when exposed to monoenergetic photons with energies of 15, 40, 50, and 68 keV. Direct and indirect single-strand breaks (SSBs), double-strand breaks (DSBs), and hybrid double-strand breaks (Hybrid DSBs) were calculated in the presence and absence of 1 to 4 nanoparticles (NPs) of the same total volume of gold, gadolinium, and silver nanoparticles for the 1ZBB model (selected from the Protein Data Bank (PDB) library). The results show that increasing the number of gold, gadolinium, and silver NPs and decreasing the photon beam energy increases the total number of strand breaks. Furthermore, gold nanoparticles (GNPs) are more effective options than gadolinium nanoparticles (GdNPs), and silver nanoparticles (SNPs) for inhibiting and controlling cancer cells. [ABSTRACT FROM AUTHOR]

Double-strand breaks (DSBs) are the most dangerous injuries for a genome. When unrepaired, death quickly ensues. In most bacterial systems, DSBs are repaired through homologous recombination. Nearly one-quarter of bacterial species harbor a second system, allowing direct ligation of broken ends, known as Non- Homologous End Joining (NHEJ). The relative role of both systems in DSBs repair in bacteria has been explored only in a few cases. To evaluate this in the bacterium Rhizobium etli, we used a modified version of the symbiotic plasmid (264kb), containing a single copy of the nifH gene. In this plasmid, we inserted an integrative plasmid harboring a modified nifH gene fragment containing an I-SceI site. DSBs were easily inflicted in vivo by conjugating a small, replicative plasmid that expresses the I-SceI nuclease into the appropriate strains. Repair of a DSB may be achieved through homologous recombination (either between adjacent or distant repeats) or NHEJ. Characterization of the derivatives that repaired DSB in different configurations, revealed that in most cases (74%), homologous recombination was the prevalent mechanism responsible for repair, with a relatively minor contribution of NHEJ (23%). Inactivation of the I-SceI gene was detected in 3% of the cases. Sequence analysis of repaired derivatives showed the operation of NHEJ. To enhance the number of derivatives repaired through NHEJ, we repeated these experiments in a recA mutant background. Derivatives showing NHEJ were readily obtained when the DSB occurred on a small, artificial plasmid in a recA mutant. However, attempts to deliver a DSB on the symbiotic plasmid in a recA background failed, due to the accumulation of mutations that inactivated the I-SceI gene. This result, coupled with the absence of derivatives that lost the nonessential symbiotic plasmid, may be due to an unusual stability of the symbiotic plasmid, possibly caused by the presence of multiple toxin-antitoxin modules. [ABSTRACT FROM AUTHOR]

Double-strand breaks (DSBs) are the most dangerous injuries for a genome. When unrepaired, death quickly ensues. In most bacterial systems, DSBs are repaired through homologous recombination. Nearly one-quarter of bacterial species harbor a second system, allowing direct ligation of broken ends, known as Non-Homologous End Joining (NHEJ). The relative role of both systems in DSBs repair in bacteria has been explored only in a few cases. To evaluate this in the bacterium Rhizobium etli, we used a modified version of the symbiotic plasmid (264 kb), containing a single copy of the nifH gene. In this plasmid, we inserted an integrative plasmid harboring a modified nifH gene fragment containing an I-SceI site. DSBs were easily inflicted in vivo by conjugating a small, replicative plasmid that expresses the I-SceI nuclease into the appropriate strains. Repair of a DSB may be achieved through homologous recombination (either between adjacent or distant repeats) or NHEJ. Characterization of the derivatives that repaired DSB in different configurations, revealed that in most cases (74%), homologous recombination was the prevalent mechanism responsible for repair, with a relatively minor contribution of NHEJ (23%). Inactivation of the I-SceI gene was detected in 3% of the cases. Sequence analysis of repaired derivatives showed the operation of NHEJ. To enhance the number of derivatives repaired through NHEJ, we repeated these experiments in a recA mutant background. Derivatives showing NHEJ were readily obtained when the DSB occurred on a small, artificial plasmid in a recA mutant. However, attempts to deliver a DSB on the symbiotic plasmid in a recA background failed, due to the accumulation of mutations that inactivated the I-SceI gene. This result, coupled with the absence of derivatives that lost the nonessential symbiotic plasmid, may be due to an unusual stability of the symbiotic plasmid, possibly caused by the presence of multiple toxin-antitoxin modules.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type-II topoisomerase-like protein that becomes covalently linked to DSB ends. Whilst Spo11 oligos—the products of nucleolytic removal by Mre11—have been detected in several organisms, the lifetime of the covalent Spo11-DSB precursor has not been determined and may be subject to alternative processing. Here, we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2—a protein known to repair DNA ends arising from abortive topoisomerase activity—on Spo11 DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 peptides from ssDNA oligos and dsDNA ends even in the presence of competitor genomic DNA. Interestingly, TDP2-processed DSB ends are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate HR at Spo11 DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro , TDP2 expression in meiotic cells was unable to remove Spo11 in vivo —contrasting its ability to aid repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher-order protein complexes at sites of meiotic recombination. • Mammalian TDP2 removes Spo11 peptides from DNA ends in vitro. • TDP2-processed ends are refractory to resection by Exo1 in vitro. • Ectopic expression of TDP2 is unable to remove Spo11 from DSB ends in vivo. [ABSTRACT FROM AUTHOR]

Encounters between replication forks and unrepaired DNA single-strand breaks (SSBs) can generate both single-ended and double-ended double-strand breaks (seDSBs and deDSBs). seDSBs can be repaired by break-induced replication (BIR), which is a highly mutagenic pathway that is thought to be responsible for many of the mutations and genome rearrangements that drive cancer development. However, the frequency of BIR's deployment and its ability to be triggered by both leading and lagging template strand SSBs were unclear. Using site- and strand-specific SSBs generated by nicking enzymes, including CRISPR-Cas9 nickase (Cas9n), we demonstrate that leading and lagging template strand SSBs in fission yeast are typically converted into deDSBs that are repaired by homologous recombination. However, both types of SSBs can also trigger BIR, and the frequency of these events increases when fork convergence is delayed and the non-homologous end joining protein Ku70 is deleted., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tuberculosis is the leading cause of mortality by infectious agents worldwide. The necrotic debris, known as caseum, which accumulates in the center of pulmonary lesions and cavities is home to nonreplicating drug-tolerant Mycobacterium tuberculosis that presents a significant hurdle to achieving a fast and durable cure. Fluoroquinolones such as moxifloxacin are highly effective at killing this nonreplicating persistent bacterial population and boosting TB lesion sterilization. Fluoroquinolones target bacterial DNA gyrase, which catalyzes the negative supercoiling of DNA and relaxes supercoils ahead of replication forks. In this study, we investigated the potency of several other classes of gyrase inhibitors against M. tuberculosis in different states of replication. In contrast to fluoroquinolones, many other gyrase inhibitors kill only replicating bacterial cultures but produce negligible cidal activity against M. tuberculosis in ex vivo rabbit caseum. We demonstrate that while these inhibitors are capable of inhibiting M. tuberculosis gyrase DNA supercoiling activity, fluoroquinolones are unique in their ability to cleave double-stranded DNA at low micromolar concentrations. We hypothesize that double-strand break formation is an important driver of gyrase inhibitor-mediated bactericidal potency against nonreplicating persistent M. tuberculosis populations in the host. This study provides general insight into the lesion sterilization potential of different gyrase inhibitor classes and informs the development of more effective chemotherapeutic options against persistent mycobacterial infections.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role. • Mutation of the KBM-binding domain in KU80 increases sensitivity to DNA replication stress • CHO cells expressing KU80 with a mutated KBM-binding domain accumulate high levels of pan-nuclear γH2AX in S/G2 phase • This phenotype is also observed in CHO cells in which the KBM-containing protein WRN is depleted. • These data implicate the KBM-binding domain in KU80 in the response to replication stress, and suggest that this role is facilitated via WRN. [ABSTRACT FROM AUTHOR]

Deinococcus radiodurans , with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system. [ABSTRACT FROM AUTHOR]

Living tissues could suffer different types of DNA damage as a result of being exposed to ionizing radiations. Monte Carlo simulations of the underlying interactions have been instrumental in predicting the damage types and the processes involved. In this work, we employed Geant4-DNA and MCDS for extracting the initial DNA damage and investigating the dependence of damage efficiency on the cell's oxygen content. The frequency-mean lineal (y¯F) and specific (z¯F) energies were derived for a spherical volume of water of various diameters between 2 and 11.1 μm. This sphere would serve as the nucleus of a cell of 100 μm diameter, engulfed by a homogeneous beam of protons. These microdosimetric quantities were calculated assuming spherical samples of 1 μm diameter in MCDS. The simulation results showed that for 230 MeV protons, an increase in the oxygen content from 0 by 10% raised the frequency of single- and double-strand breaks and lowered the base damage frequency. The resulting damage frequencies appeared to be independent of nucleus diameter. For proton energies between 2 and 230 MeV,y¯Fshowed no dependence on the cell diameter and an increase of the cell size resulted in a decrease inz¯F.An increase in the proton energy slowed down the decreasing rate ofz¯Fas a function of nucleus diameter. However, the ratio ofy¯Fvalues corresponding to two proton energies of choice showed no dependence on the nucleus size and were equal to the ratio of the correspondingz¯Fvalues. Furthermore, the oxygen content of the cell did not affect these microdosimetric quantities. Contrary to damage frequencies, these quantities appeared to depend only on direct interactions due to deposited energies. Our calculations showed the near independence of DNA damages on the nucleus size of the human cells. The probabilities of different types of single and double-strand breaks increase with the oxygen content., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we found that in P - M hybrid dysgenesis in Drosophila , a sterility syndrome triggered by the P -element DNA transposon, germ cells harbor unexpectedly few new TE insertions despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P -elements or other noncoding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development., (© 2024 Jansen et al.; Published by Cold Spring Harbor Laboratory Press.)

CRISPR-Cas systems revolutionized the genome engineering field but need to induce double-strand breaks (DSBs) and may be difficult to deliver due to their large protein size. Tn7-like transposons such as CRISPR-associated transposons (CASTs) can be repurposed for RNA-guided DSB-free integration, and obligate mobile element guided activity (OMEGA) proteins of the IS200/IS605 transposon family have been developed as hypercompact RNA-guided genome editing tools. CASTs and OMEGA are exciting, innovative genome engineering tools that can improve the precision and efficiency of editing. This review explores the recent developments and uses of CASTs and OMEGA in genome editing across prokaryotic and eukaryotic cells. The pros and cons of these transposon-based systems are deliberated in comparison to other CRISPR systems., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Radiotherapy (RT) is used for over 50 % of cancer patients and can promote adaptive immunity against tumour antigens. However, the underlying mechanisms remain unclear. Here, we discovered that RT induces the release of irradiated tumour cell-derived microparticles (RT-MPs), which significantly upregulate MHC-I expression on the membranes of non-irradiated cells, enhancing the recognition and killing of these cells by T cells. Mechanistically, RT-MPs induce DNA double-strand breaks (DSB) in tumour cells, activating the ATM/ATR/CHK1-mediated DNA repair signalling pathway, and upregulating MHC-I expression. Inhibition of ATM/ATR/CHK1 reversed RT-MP-induced upregulation of MHC-I. Furthermore, phosphorylation of STAT1/3 following the activation of ATM/ATR/CHK1 is indispensable for the DSB-dependent upregulation of MHC-I. Therefore, our findings reveal the role of RT-MP-induced DSBs and the subsequent DNA repair signalling pathway in MHC-I expression and provide mechanistic insights into the regulation of MHC-I expression after DSBs. • Irradiated tumor cell-derived microparticles (RT-MPs) upregulate MHC-I expression, marking the first evidence of radiation-induced bystander effect (RIBE) in this context. • RT-MPs enhance CD8+ T cell-mediated antigen-specific recognition and subsequent killing of tumour cells. • RT-MPs induce double-stand breaks in tumour cells. • Inhibition of ATM/ATR/CHK1 reversed RT-MP induced MHC-I expression. [ABSTRACT FROM AUTHOR]

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1 -deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1. [Display omitted] • Loss of 53BP1 increases HDR and reprogramming efficiency in mouse and human cells • Brca1 phospho-interaction with Abraxas, Bach1, CtIP is required for HDR and reprogramming • Stalled replication fork protection by Brca1 is dispensable for reprogramming • Brca2 mutants with replication gap suppression deficiency reprogram efficiently Using separation-of-function mutations in Brca1, Brca2, and interacting proteins, we show that homology-directed repair of replication-associated double -strand breaks is the critical function of the BRCA pathway in iPSC generation. Stalled replication fork protection and replication gap suppression are inconsequential to efficient iPSC induction. [ABSTRACT FROM AUTHOR]

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 early meiotic prophase ensuring that SPO11 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 coordinates the actions of the different accessory factors sub-groups, providing insights into how components on the DNA loops may interact with the chromosome axis.

Nonspecific structural chromosomal aberrations (CAs) are found in around 1% of circulating lymphocytes from healthy individuals but the frequency may be higher after exposure to carcinogenic chemicals or radiation. CAs have been used in the monitoring of persons exposed to genotoxic agents and radiation. Previous studies on occupationally exposed individuals have shown associations between the frequency of CAs in peripheral blood lymphocytes and subsequent cancer risk. The cause for CA formation are believed to be unrepaired or insufficiently repaired DNA double-strand breaks or other DNA damage, and additionally telomere shortening. CAs include chromosome (CSAs) and chromatid type aberrations (CTAs). In the present review, we first describe the types of CAs, the conventional techniques used for their detection and some aspects of interpreting the results. We then focus on germline genetic variation in the frequency and type of CAs measured in a genome-wide association study (GWAS) in healthy individuals in relation to occupational and smoking-related exposure compared to non-exposed referents. The associations (at p<10-5) on 1473 healthy individuals were broadly classified in candidate genes from functional pathways related to DNA damage response/repair, including PSMA1, UBR5, RRM2B, PMS2P4, STAG3L4, BOD1, COPRS and FTO; another group included genes related to apoptosis, cell proliferation, angiogenesis and tumorigenesis, COPB1, NR2C1, COPRS, RHOT1, ITGB3, SYK, and SEMA6A; a third small group mapped to genes KLF7, SEMA5A and ITGB3 which were related to autistic traits, known to manifest frequent CAs. Dedicated studies on 153 DNA repair genes showed associations for some 30 genes, expression of which could be modified by the implicated variants. We finally point out that monitoring of CAs is so far the only method of assessing cancer risk in healthy human populations, and the use of the technology should be made more attractive by developing automated performance steps and incorporating artificial intelligence methods into the scoring., (© The Author(s) 2024. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Immunofluorescent staining is commonly used to generate images to characterize cytological phenotypes. The manual quantification of DNA double-strand breaks and their repair intermediates during meiosis using image data requires a series of subjective steps, from image selection to the counting of particular events per nucleus. Here we describe "synapsis," a bioconductor package, which includes a set of functions to automate the process of identifying meiotic nuclei and quantifying key double-strand break formation and repair events in a rapid, scalable, and reproducible workflow, and compare it to manual user quantification. The software can be extended for other applications in meiosis research, such as incorporating machine learning approaches to categorize meiotic substages., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The relatively recent discovery of CRISPR/Cas has led to a revolution in our ability to efficiently manipulate the genome of eukaryotic cells. We describe here a protocol that employs CRISPR technology to precisely knock-in a PET imaging reporter transgene into a specific genetic locus of interest. Resulting transcription of the targeted reporter will more accurately mimic physiologic expression of the endogenous allele than conventional approaches, and so this method has the potential to become an efficient way to generate a new generation of "gold-standard" reporter transgenes. We break down the protocol into three experimental stages: how to identify the genomic location that the reporter transgene will be inserted, how to practically insert the reporter transgene into the genome, and how to screen resultant clones for the correct targeted event., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)