With the advancement of CRISPR technologies, a comprehensive understanding of repair mechanisms following double-strand break (DSB) formation is important for improving the precision and efficiency of genetic modifications. In plant genetics, two Cas nucleases are widely used, i.e. Cas9 and Cas12a, which differ with respect to PAM sequence composition, position of the DSB relative to the PAM, and DSB-end configuration (blunt vs. staggered). The latter difference has led to speculations about different options for repair and recombination. Here, we provide detailed repair profiles for LbCas12a in Arabidopsis thaliana, using identical experimental settings previously reported for Cas9-induced DSBs, thus allowing for a quantitative comparison of both nucleases. For both enzymes, non-homologous end-joining (NHEJ) produces 70% of mutations, whereas polymerase theta-mediated end-joining (TMEJ) generates 30%, indicating that DSB-end configuration does not dictate repair pathway choice. Relevant for genome engineering approaches aimed at integrating exogenous DNA, we found that Cas12a similarly stimulates the integration of T-DNA molecules as does Cas9. Long-read sequencing of both Cas9 and Cas12a repair outcomes further revealed a previously underappreciated degree of DNA loss upon TMEJ. The most notable disparity between Cas9 and Cas12a repair profiles is caused by how NHEJ acts on DSB ends with short overhangs: non-symmetric Cas9 cleavage produce 1 bp insertions, which we here show to depend on polymerase Lambda, whereas staggered Cas12a DSBs are not subjected to fill-in synthesis. We conclude that Cas9 and Cas12a are equally effective for genome engineering purposes, offering flexibility in nuclease choice based on the availability of compatible PAM sequences.

UNASSIGNED: Ewing sarcoma is an aggressive mesenchymal malignancy commonly affecting children and young adolescents. The molecular basis of this neoplasia is well reported with the formation of the EWSR1/FLI1 fusion gene being the most common genetic finding. However, this fusion gene has not been targeted therapeutically nor is being used as a prognostic marker. Its relevance regarding the molecular steps leading to Ewing sarcoma genesis are yet to be defined. The generation of the oncogenic EWSR1/FLI1 fusion gene, can be attributed to the simultaneous introduction of two DNA double-strand breaks (DSBs). The scope of this study is to detect any association between DNA repair deficiency and the clinicopathological aspects of Ewing\'s sarcoma disease.
UNASSIGNED: We have conducted an expression analysis of 35 patients diagnosed with Ewing sarcoma concerning the genes involved in non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways. We have analyzed the expression levels of 6 genes involved in NHEJ (XRCC4, XRCC5, XRCC6, POLλ, POLμ) and 9 genes involved in HR (RAD51, RAD52, RAD54, BRCA1, BRCA2, FANCC, FANCD, DNTM1, BRIT1) using real time PCR. Age, sex, location of primary tumor, tumor size, KI67, mitotic count, invasion of adjacent tissues and treatment were the clinicopathological parameters included in the statistical analysis.
UNASSIGNED: Our results show that both these DNA repair pathways are deregulated in Ewing sarcoma. In addition, low expression of the xrcc4 gene has been associated with better overall survival probability (p=0.032).
UNASSIGNED: Our results, even though retrospective and in a small number of patients, highlight the importance of DSBs repair and propose a potential therapeutic target for this type of sarcoma.

We explore the possibility that defects in genes associated with the response and repair of DNA double strand breaks predispose oral potentially malignant disorders (OPMD) to undergo malignant transformation to oral squamous cell carcinoma (OSCC). Defects in the homologous recombination/Fanconi anemia (HR/FA), but not in the non-homologous end joining, causes the DNA repair pathway to appear to be consistent with features of familial conditions that are predisposed to OSCC (FA, Bloom\'s syndrome, Ataxia Telangiectasia); this is true for OSCC that occurs in young patients, sometimes with little/no exposure to classical risk factors. Even in Dyskeratosis Congenita, a disorder of the telomerase complex that is also predisposed to OSCC, attempts at maintaining telomere length involve a pathway with shared HR genes. Defects in the HR/FA pathway therefore appear to be pivotal in conditions that are predisposed to OSCC. There is also some evidence that abnormalities in the HR/FA pathway are associated with malignant transformation of sporadic cases OPMD and OSCC. We provide data showing overexpression of HR/FA genes in a cell-cycle-dependent manner in a series of OPMD-derived immortal keratinocyte cell lines compared to their mortal counterparts. The observations in this study argue strongly for an important role of the HA/FA DNA repair pathway in the development of OSCC.

The correct repair of DNA Double Strand Breaks (DSBs) is fundamental to prevent the loss of genetic information, mutations, and chromosome rearrangements. An emerging determinant of DNA repair is chromatin mobility. However, how chromatin mobility can influence DSBs repair is still poorly understood. While increased mobility is generally associated with the correct repair by Homologous Recombination (HR) of DSBs generated in heterochromatin, it promotes the mis-repair of multiple distal DSBs by Non-Homologous End Joining (NHEJ). Here we describe a method for detecting and quantifying DSBs mobility by live-cell imaging in the context of multiple DSBs prone to mis-repair by NHEJ. In addition, we discuss a set of parameters that can be used for quantitative and qualitative analysis of nuclear deformations and to discard nuclei where the deformation could affect the analysis of DSBs mobility. While this method is based on the visualization of DSBs with the mCherry-53BP1-2 fusion protein, we believe that it can also be used to analyze the mobility of nuclear foci formed by different fluorescent proteins.

The glycocalyx-that forms a protective barrier around cells-has been implicated in cancer cell proliferation, survival, and metastasis. However, its role in maintaining the integrity of DNA/nucleus during migration through dense matrices remains unexplored. In this study, we address this question by first documenting heterogeneity in glycocalyx expression in highly invasive MDA-MB-231 breast cancer cells, and establishing a negative correlation between cell size and glycocalyx levels. Next, we set-up transwell migration through 3 µm pores, to isolate two distinct sub-populations and to show that the early migrating cell sub-population possesses a bulkier glycocalyx and undergoes less DNA damage and nuclear rupture, assessed using γH2AX foci formation and nuclear/cytoplasmic distribution of Ku70/80. Interestingly, enzymatic removal of glycocalyx led to disintegration of the nuclear membrane indicated by increased cytoplasmic localisation of Ku70/80, increased nuclear blebbing and reduction in nuclear area. Together, these results illustrate an inverse association between bulkiness of the glycocalyx and nuclear stresses, and highlights the mechanical role of the glycocalyx in shielding migration associated stresses.

Although mechanisms of telomere protection are well-defined in differentiated cells, it is poorly understood how stem cells sense and respond to telomere dysfunction. In particular, the broader impact of telomeric double-strand breaks (DSBs) in these cells is poorly characterized. Here, we report on DNA damage signaling, cell cycle, and transcriptome-level changes in human induced pluripotent stem cells (iPSCs) in response to telomere-internal DSBs. We engineered human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres. Using this model, we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DDR, which leads to p53-independent cell cycle arrest in G2. Using CRISPR-Cas9 to cripple the catalytic domain of telomerase, we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres, which instead are effectively repaired by robust homologous recombination (HR). In contrast to HR-based telomere maintenance in mouse embryonic stem cells, we find neither evidence that HR causes extension of telomeres beyond their initial lengths, nor an apparent role for ZSCAN4 in this process. Rather, HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length which is compatible with sustained survival of the cells over several days of TRF1-FokI induction. Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric damage.

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11β splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11βki/- mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11βki/- males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11β and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.

XLF/Cernunnos is a component of the ligation complex used in classical non-homologous end-joining (cNHEJ), a major DNA double-strand break (DSB) repair pathway. We report neurodevelopmental delays and significant behavioral alterations associated with microcephaly in Xlf-/- mice. This phenotype, reminiscent of clinical and neuropathologic features in humans deficient in cNHEJ, is associated with a low level of apoptosis of neural cells and premature neurogenesis, which consists of an early shift of neural progenitors from proliferative to neurogenic divisions during brain development. We show that premature neurogenesis is related to an increase in chromatid breaks affecting mitotic spindle orientation, highlighting a direct link between asymmetric chromosome segregation and asymmetric neurogenic divisions. This study reveals thus that XLF is required for maintaining symmetric proliferative divisions of neural progenitors during brain development and shows that premature neurogenesis may play a major role in neurodevelopmental pathologies caused by NHEJ deficiency and/or genotoxic stress.

We present an investigation of the effects on BxPC3 pancreatic cancer cells of proton therapy combined with hyperthermia, assisted by magnetic fluid hyperthermia performed with the use of magnetic nanoparticles. The cells\' response to the combined treatment has been evaluated by means of the clonogenic survival assay and the estimation of DNA Double Strand Breaks (DSBs). The Reactive Oxygen Species (ROS) production, the tumor cell invasion and the cell cycle variations have also been studied. The experimental results have shown that the combination of proton therapy, MNPs administration and hyperthermia gives a clonogenic survival that is much smaller than the single irradiation treatment at all doses, thus suggesting a new effective combined therapy for the pancreatic tumor. Importantly, the effect of the therapies used here is synergistic. Moreover, after proton irradiation, the hyperthermia treatment was able to increase the number of DSBs, even though just at 6 h after the treatment. Noticeably, the magnetic nanoparticles\' presence induces radiosensitization effects, and hyperthermia increases the production of ROS, which contributes to cytotoxic cellular effects and to a wide variety of lesions including DNA damage. The present study indicates a new way for clinical translation of combined therapies, also in the vision of an increasing number of hospitals that will use the proton therapy technique in the near future for different kinds of radio-resistant cancers.

Liver cirrhosis and hepatocellular carcinoma are the most common outcomes of chronic hepatitis B. Hepatitis B virus (HBV) induces transformation and cell death in chronic hepatitis B (CHB). DNA double strand breaks (DSBs) represent the most dangerous type of genome damage. It was shown previously that generation of phosphorylated histone H2AX foci is a reliable marker of DSBs. The aim of this study was to analyse generation of yH2AX foci in HBV and hepatitis D virus (HDV) infection in vitro and in liver biopsies of patients with CHB and CHB with delta-agent (CHD). Human hepatoma cell line HepG2-1.1merHBV with activated HBV life cycle was used to perform real-time PCR for analysis of pregenomic RNA, HBV DNA, HBV cccDNA and for immunocytochemical analysis of yH2AX. Liver biopsies from CHB and CHD patients were analyzed to confirm the results. HBV induces multiple discrete yH2AX foci in HepG2-1.1merHBV cells in vitro and in biopsies of CHB and CHB+D patients. The ratio of hepatocytes w/o yH2AX foci is significantly lower (49,9+/-12,3% vs. 85,5+/-0,9%, p.
Исходами хронического гепатита В (ХГВ) являются цирроз печени и гепатоцеллюлярная карцинома. Гибель и трансформация гепатоцитов при ХГВ связаны с влиянием вируса гепатита В (HBV) на клетку. Самым опасным видом повреждения генома клеток является образование двухцепочечных разрывов ДНК (ДЦР). Ранее было показано, что образование фокусов фосфорилированного гистона Н2АХ (уН2АХ) является надёжным индикатором ДЦР. Целью работы было изучение формирования фокусов уН2АХ при инфекции, вызванной HBV и HDV, на модели HBV in vitro, а также в биоптатах пациентов с ХГВ и ХГВ с дельта-агентом в биоптатах печени. Клетки гепатомы человека HepG2-1.1merHBV с активным циклом HBV были использованы для оценки экспрессии прегеномной РНК, уровней ДНК и кольцевой ковалентно-замкнутой ДНК (ккзДНК) HBV и иммуноцитохимического анализа образования yH2AX-гистона. Срезы биоптатов печени пациентов с ХГВ и ХГВ + D использовали для подтверждения результатов по генерации уН2АХ-гистона. В результате показано, что HBV вызывает образование многочисленных фокусов уН2АХ в культуре клеток HepG2-1.1merHBV in vitro и гепатоцитах пациентов с ХГВ и ХГB + D. В гепатоцитах пациента с ХГВ доля клеток без фокусов значительно ниже (49,9 ± 12,3% против 85,5 ± 0,9%; p < 0,05), а доля клеток с 1-10 фокусами уН2АХ выше (49,3 ± 12,6% против 14,5 ± 0,9%; p < 0,05) в сравнении со здоровым донором. При ХГВ + D происходит увеличение среднего числа уН2АХ-фокусов (3,5 ± 1,1 и 5,5 ± 1,5 против 0,5 ± 0,16 в контроле; p < 0,05). У пациентов c ХГВ и ХГВ + D снижается доля гепатоцитов без уН2АХ, возрастает доля клеток с 1-10 уН2АХ, появляются клетки с многочисленными (11-30 уН2АХ/клетку) фокусами. Таким образом, yH2AX-фокусы образуются при инфекции HBV in vitro, в гепатоцитах пациентов с ХГВ и ХГB + D и могут использоваться для оценки повреждения генома, связанного с HBV и HDV.