The crucial role of HFM1 in regulating FUS ubiquitination and localization for oocyte meiosis prophase I progression in mice.

BACKGROUND: Helicase for meiosis 1 (HFM1), a putative DNA helicase expressed in germ-line cells, has been reported to be closely associated with premature ovarian insufficiency (POI). However, the underlying molecular mechanism has not been clearly elucidated. The aim of this study was to investigate the function of HFM1 in the first meiotic prophase of mouse oocytes. RESULTS: The results suggested that the deficiency of HFM1 resulting in increased apoptosis and depletion of oocytes in mice, while the oocytes were arrested in the pachytene stage of the first meiotic prophase. In addition, impaired DNA double-strand break repair and disrupted synapsis were observed in the absence of HFM1. Further investigation revealed that knockout of HFM1 promoted ubiquitination and degradation of FUS protein mediated by FBXW11. Additionally, the depletion of HFM1 altered the intranuclear localization of FUS and regulated meiotic- and oocyte development-related genes in oocytes by modulating the expression of BRCA1. CONCLUSIONS: These findings elaborated that the critical role of HFM1 in orchestrating the regulation of DNA double-strand break repair and synapsis to ensure meiosis procession and primordial follicle formation. This study provided insights into the pathogenesis of POI and highlighted the importance of HFM1 in maintaining proper meiotic function in mouse oocytes.

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

Nono induces Gadd45b to mediate DNA repair.

RNA-binding proteins are frequently deregulated in cancer and emerge as effectors of the DNA damage response (DDR). The non-POU domain-containing octamer-binding protein NONO/p54nrb is a multifunctional RNA-binding protein that not only modulates the production and processing of mRNA, but also promotes the repair of DNA double-strand breaks (DSBs). Here, we investigate the impact of Nono deletion in the murine KP (KRas G12D , Trp53 -/- ) cell-based lung cancer model. We show that the deletion of Nono impairs the response to DNA damage induced by the topoisomerase II inhibitor etoposide or the radiomimetic drug bleomycin. Nono-deficient KP (KPN) cells display hyperactivation of DSB signalling and high levels of DSBs. The defects in the DDR are accompanied by reduced RNA polymerase II promoter occupancy, impaired nascent RNA synthesis, and attenuated induction of the DDR factor growth arrest and DNA damage-inducible beta (Gadd45b). Our data characterise Gadd45b as a putative Nono-dependent effector of the DDR and suggest that Nono mediates a genome-protective crosstalk of the DDR with the RNA metabolism via induction of Gadd45b.

RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair.

RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.

Radiation-induced DNA double-strand breaks in cortisol exposed fibroblasts as quantified with the novel foci-integrated damage complexity score (FIDCS).

Without the protective shielding of Earth's atmosphere, astronauts face higher doses of ionizing radiation in space, causing serious health concerns. Highly charged and high energy (HZE) particles are particularly effective in causing complex and difficult-to-repair DNA double-strand breaks compared to low linear energy transfer. Additionally, chronic cortisol exposure during spaceflight raises further concerns, although its specific impact on DNA damage and repair remains unknown. This study explorers the effect of different radiation qualities (photons, protons, carbon, and iron ions) on the DNA damage and repair of cortisol-conditioned primary human dermal fibroblasts. Besides, we introduce a new measure, the Foci-Integrated Damage Complexity Score (FIDCS), to assess DNA damage complexity by analyzing focus area and fluorescent intensity. Our results show that the FIDCS captured the DNA damage induced by different radiation qualities better than counting the number of foci, as traditionally done. Besides, using this measure, we were able to identify differences in DNA damage between cortisol-exposed cells and controls. This suggests that, besides measuring the total number of foci, considering the complexity of the DNA damage by means of the FIDCS can provide additional and, in our case, improved information when comparing different radiation qualities.

PARP1-DNA co-condensation: the driver of broken DNA repair.

DNA double-strand break (DSB) sites that prevent the disjunction of broken DNA ends are formed through poly (ADP-ribose) (PAR) polymerase 1 (PARP1)-DNA co-condensation. The co-condensates apply mechanical forces to hold the DNA ends together and generate enzymatic activity for the synthesis of PAR. PARylation can promote the release of PARP1 from DNA ends and recruit various proteins, such as Fused in sarcoma (FUS) proteins, thereby stabilizing broken DNA ends and preventing their separation.

MYB4, a member of R2R3-subfamily of MYB transcription factor functions as a repressor of key genes involved in flavonoid biosynthesis and repair of UV-B induced DNA double strand breaks in Arabidopsis.

Plants accumulate flavonoids as part of UV-B acclimation, while a high level of UV-B irradiation induces DNA damage and leads to genome instability. Here, we show that MYB4, a member of the R2R3-subfamily of MYB transcription factor plays important role in regulating plant response to UV-B exposure through the direct repression of the key genes involved in flavonoids biosynthesis and repair of DNA double-strand breaks (DSBs). Our results demonstrate that MYB4 inhibits seed germination and seedling establishment in Arabidopsis following UV-B exposure. Phenotype analyses of atmyb4-1 single mutant line along with uvr8-6/atmyb4-1, cop1-6/atmyb4-1, and hy5-215/atmyb4-1 double mutants indicate that MYB4 functions downstream of UVR8 mediated signaling pathway and negatively affects UV-B acclimation and cotyledon expansion. Our results indicate that MYB4 acts as transcriptional repressor of two key flavonoid biosynthesis genes, including 4CL and FLS, via directly binding to their promoter, thus reducing flavonoid accumulation. On the other hand, AtMYB4 overexpression leads to higher accumulation level of DSBs along with repressed expression of several key DSB repair genes, including AtATM, AtKU70, AtLIG4, AtXRCC4, AtBRCA1, AtSOG1, AtRAD51, and AtRAD54, respectively. Our results further suggest that MYB4 protein represses the expression of two crucial DSB repair genes, AtKU70 and AtXRCC4 through direct binding with their promoters. Together, our results indicate that MYB4 functions as an important coordinator to regulate plant response to UV-B through transcriptional regulation of key genes involved in flavonoids biosynthesis and repair of UV-B induced DNA damage.

Molecular and cellular consequences of mitochondrial DNA double-stranded breaks.

Mitochondria are subcellular organelles essential for life. Beyond their role in producing energy, mitochondria govern various physiological mechanisms, encompassing energy generation, metabolic processes, apoptotic events, and immune responses. Mitochondria also contain genetic material that is susceptible to various forms of damage. Mitochondrial double-stranded breaks (DSB) are toxic lesions that the nucleus repairs promptly. Nevertheless, the significance of DSB repair in mammalian mitochondria is controversial. This review presents an updated view of the available research on the consequences of mitochondrial DNA DSB from the molecular to the cellular level. We discuss the crucial function of mitochondrial DNA damage in regulating processes such as senescence, integrated stress response, and innate immunity. Lastly, we discuss the potential role of mitochondrial DNA DSB in mediating the cellular consequences of ionizing radiations, the standard of care in treating solid tumors.

Synthesis and new DNA targeting activity of 6- and 7-tert-butylfascaplysins.

Fascaplysin is a red cytotoxic pigment with anticancer properties isolated from the marine sponge Fascaplysinopsis sp. Recently, structure-activity relationship analysis reported by our group suggested that selective cytotoxicity of fascaplysin derivatives towards tumor cells negatively correlates with their ability to intercalate into DNA. To validate this hypothesis, we synthesized 6- and 7-tert-butylfascaplysins which reveal mitigated DNA-intercalating properties. These derivatives were found to be strongly cytotoxic to drug-resistant human prostate cancer cells, albeit did not demonstrate improved selectivity towards cancer cells when compared to fascaplysin. At the same time, kinome analysis suggested an activation of CHK1/ATR axis in cancer cells shortly after the drug exposure. Further experiments revealed induction of replication stress that is eventually converted to the toxic DNA double-strand breaks, resulting in caspase-independent apoptosis-like cell death. Our observations highlight new DNA-targeting effect of some fascaplysin derivatives and indicate more complex structure-activity relationships within the fascaplysin family, suggesting that cytotoxicity and selectivity of these alkaloids are influenced by multiple factors. Furthermore, combination with clinically-approved inhibitors of ATR/CHK1 as well as testing in tumors particularly sensitive to the DNA damage should be considered in further studies.

Induced Pluripotent Stem Cell-Derived Fibroblasts Efficiently Engage Senescence Pathways but Show Increased Sensitivity to Stress Inducers.

The risk of aberrant growth of induced pluripotent stem cell (iPSC)-derived cells in response to DNA damage is a potential concern as the tumor suppressor genes TP53 and CDKN2A are transiently inactivated during reprogramming. Herein, we evaluate the integrity of cellular senescence pathways and DNA double-strand break (DSB) repair in Sendai virus reprogrammed iPSC-derived human fibroblasts (i-HF) compared to their parental skin fibroblasts (HF). Using transcriptomics analysis and a variety of functional assays, we show that the capacity of i-HF to enter senescence and repair DSB is not compromised after damage induced by ionizing radiation (IR) or the overexpression of H-RASV12. Still, i-HF lines are transcriptionally different from their parental lines, showing enhanced metabolic activity and higher expression of p53-related effector genes. As a result, i-HF lines generally exhibit increased sensitivity to various stresses, have an elevated senescence-associated secretory phenotype (SASP), and cannot be immortalized unless p53 expression is knocked down. In conclusion, while our results suggest that i-HF are not at a greater risk of transformation, their overall hyperactivation of senescence pathways may impede their function as a cell therapy product.

RNA polymerase II-mediated rDNA transcription mediates rDNA copy number expansion in Drosophila.

Ribosomal DNA (rDNA), which encodes ribosomal RNA, is an essential but unstable genomic element due to its tandemly repeated nature. rDNA's repetitive nature causes spontaneous intrachromatid recombination, leading to copy number (CN) reduction, which must be counteracted by a mechanism that recovers CN to sustain cells' viability. Akin to telomere maintenance, rDNA maintenance is particularly important in cell types that proliferate for an extended time period, most notably in the germline that passes the genome through generations. In Drosophila, the process of rDNA CN recovery, known as 'rDNA magnification', has been studied extensively. rDNA magnification is mediated by unequal sister chromatid exchange (USCE), which generates a sister chromatid that gains the rDNA CN by stealing copies from its sister. However, much remains elusive regarding how germ cells sense rDNA CN to decide when to initiate magnification, and how germ cells balance between the need to generate DNA double-strand breaks (DSBs) to trigger USCE vs. avoiding harmful DSBs. Recently, we identified an rDNA-binding Zinc-finger protein Indra as a factor required for rDNA magnification, however, the underlying mechanism of action remains unknown. Here we show that Indra is a negative regulator of rDNA magnification, balancing the need of rDNA magnification and repression of dangerous DSBs. Mechanistically, we show that Indra is a repressor of RNA polymerase II (Pol II)-dependent transcription of rDNA: Under low rDNA CN conditions, Indra protein amount is downregulated, leading to Pol II-mediated transcription of rDNA. This results in the expression of rDNA-specific retrotransposon, R2, which we have shown to facilitate rDNA magnification via generation of DBSs at rDNA. We propose that differential use of Pol I and Pol II plays a critical role in regulating rDNA CN expansion only when it is necessary.

Modelling the In Vivo and Ex Vivo DNA Damage Response after Internal Irradiation of Blood from Patients with Thyroid Cancer.

This work reports on a model that describes patient-specific absorbed dose-dependent DNA damage response in peripheral blood mononuclear cells of thyroid cancer patients during radioiodine therapy and compares the results with the ex vivo DNA damage response in these patients. Blood samples of 18 patients (nine time points up to 168 h post-administration) were analyzed for radiation-induced γ-H2AX + 53BP1 DNA double-strand break foci (RIF). A linear one-compartment model described the absorbed dose-dependent time course of RIF (Parameters: c characterizes DSB damage induction; k1 and k2 are rate constants describing fast and slow repair). The rate constants were compared to ex vivo repair rates. A total of 14 patient datasets could be analyzed; c ranged from 0.012 to 0.109 mGy-1, k2 from 0 to 0.04 h-1. On average, 96% of the damage is repaired quickly with k1 (range: 0.19-3.03 h-1). Two patient subgroups were distinguished by k1-values (n = 6, k1 > 1.1 h-1; n = 8, k1 < 0.6 h-1). A weak correlation with patient age was observed. While induction of RIF was similar among ex vivo and in vivo, the respective repair rates failed to correlate. The lack of correlation between in vivo and ex vivo repair rates and the applicability of the model to other therapies will be addressed in further studies.

TDP1 mutation causing SCAN1 neurodegenerative syndrome hampers the repair of transcriptional DNA double-strand breaks.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

RadPhysBio: A Radiobiological Database for the Prediction of Cell Survival upon Exposure to Ionizing Radiation.

Based on the need for radiobiological databases, in this work, we mined experimental ionizing radiation data of human cells treated with X-rays, γ-rays, carbon ions, protons and α-particles, by manually searching the relevant literature in PubMed from 1980 until 2024. In order to calculate normal and tumor cell survival α and ß coefficients of the linear quadratic (LQ) established model, as well as the initial values of the double-strand breaks (DSBs) in DNA, we used WebPlotDigitizer and Python programming language. We also produced complex DNA damage results through the fast Monte Carlo code MCDS in order to complete any missing data. The calculated α/ß values are in good agreement with those valued reported in the literature, where α shows a relatively good association with linear energy transfer (LET), but not ß. In general, a positive correlation between DSBs and LET was observed as far as the experimental values are concerned. Furthermore, we developed a biophysical prediction model by using machine learning, which showed a good performance for α, while it underscored LET as the most important feature for its prediction. In this study, we designed and developed the novel radiobiological 'RadPhysBio' database for the prediction of irradiated cell survival (α and ß coefficients of the LQ model). The incorporation of machine learning and repair models increases the applicability of our results and the spectrum of potential users.

DNA damage response in a 2D-culture model by diffusing alpha-emitters radiation therapy (Alpha-DaRT).

Diffusing alpha-emitters radiation therapy (Alpha-DaRT) is a unique method, in which interstitial sources carrying 224Ra release a chain of short-lived daughter atoms from their surface. Although DNA damage response (DDR) is crucial to inducing cell death after irradiation, how the DDR occurs during Alpha-DaRT treatment has not yet been explored. In this study, we temporo-spatially characterized DDR such as kinetics of DNA double-strand breaks (DSBs) and cell cycle, in two-dimensional (2D) culture conditions qualitatively mimicking Alpha-DaRT treatments, by employing HeLa cells expressing the Fucci cell cycle-visualizing system. The distribution of the alpha-particle pits detected by a plastic nuclear track detector, CR-39, strongly correlated with γH2AX staining, a marker of DSBs, around the 224Ra source, but the area of G2 arrested cells was more widely spread 24 h from the start of the exposure. Thereafter, close time-lapse observation revealed varying cell cycle kinetics, depending on the distance from the source. A medium containing daughter nuclides prepared from 224Ra sources allowed us to estimate the radiation dose after 24 h of exposure, and determine surviving fractions. The present experimental model revealed for the first time temporo-spatial information of DDR occurring around the source in its early stages.

BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

Bridging the gap between the evolutionary dynamics and the molecular mechanisms of meiosis: A model based exploration of the PRDM9 intra-genomic Red Queen.

Molecular dissection of meiotic recombination in mammals, combined with population-genetic and comparative studies, have revealed a complex evolutionary dynamic characterized by short-lived recombination hotspots. Hotspots are chromosome positions containing DNA sequences where the protein PRDM9 can bind and cause crossing-over. To explain these fast evolutionary dynamic, a so-called intra-genomic Red Queen model has been proposed, based on the interplay between two antagonistic forces: biased gene conversion, mediated by double-strand breaks, resulting in hotspot extinction (the hotspot conversion paradox), followed by positive selection favoring mutant PRDM9 alleles recognizing new sequence motifs. Although this model predicts many empirical observations, the exact causes of the positive selection acting on new PRDM9 alleles is still not well understood. In this direction, experiment on mouse hybrids have suggested that, in addition to targeting double strand breaks, PRDM9 has another role during meiosis. Specifically, PRDM9 symmetric binding (simultaneous binding at the same site on both homologues) would facilitate homology search and, as a result, the pairing of the homologues. Although discovered in hybrids, this second function of PRDM9 could also be involved in the evolutionary dynamic observed within populations. To address this point, here, we present a theoretical model of the evolutionary dynamic of meiotic recombination integrating current knowledge about the molecular function of PRDM9. Our modeling work gives important insights into the selective forces driving the turnover of recombination hotspots. Specifically, the reduced symmetrical binding of PRDM9 caused by the loss of high affinity binding sites induces a net positive selection eliciting new PRDM9 alleles recognizing new targets. The model also offers new insights about the influence of the gene dosage of PRDM9, which can paradoxically result in negative selection on new PRDM9 alleles entering the population, driving their eviction and thus reducing standing variation at this locus.

Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα: Dynophore-derived discovery.

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

Microhomology-mediated repair machinery and its relationship with HPV-mediated oncogenesis.

Human Papillomaviruses (HPV) are a diverse family of non-enveloped dsDNA viruses that infect the skin and mucosal epithelia. Persistent HPV infections can lead to cancer frequently involving integration of the virus into the host genome, leading to sustained oncogene expression and loss of capsid and genome maintenance proteins. Microhomology-mediated double-strand break repair, a DNA double-stranded breaks repair pathway present in many organisms, was initially thought to be a backup but it's now seen as vital, especially in homologous recombination-deficient contexts. Increasing evidence has identified microhomology (MH) near HPV integration junctions, suggesting MH-mediated repair pathways drive integration. In this comprehensive review, we present a detailed summary of both the mechanisms underlying MH-mediated repair and the evidence for its involvement in HPV integration in cancer. Lastly, we highlight the involvement of these processes in the integration of other DNA viruses and the broader implications on virus lifecycles and host innate immune response.