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.

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.

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.

Fusion of histone variants to Cas9 suppresses non-homologous end joining.

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

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.

Dose and DNA damage modelling of diffusing alpha-emitters radiation therapy using Geant4.

PURPOSE: Diffusing alpha-emitters radiation therapy (DaRT) is a brachytherapy technique using α-particles to treat solid tumours. The high linear energy transfer (LET) and short range of α-particles make them good candidates for the targeted treatment of cancer. Treatment planning of DaRT requires a good understanding of the dose from α-particles and the other particles released in the 224Ra decay chain. METHODS: The Geant4 Monte Carlo toolkit has been used to simulate a DaRT seed to better understand the dose contribution from all particles and simulate the DNA damage due to this treatment. RESULTS: Close to the seed α-particles deliver the majority of dose, however at radial distances greater than 4 mm, the contribution of ß-particles is greater. The RBE has been estimated as a function of number of double strand breaks (DSBs) and complex DSBs. A maximum seed spacing of 5.5 mm and 6.5 mm was found to deliver at least 20 Gy RBE weighted dose between the seeds for RBEDSB and RBEcDSB respectively. CONCLUSIONS: The DNA damage changes with radial distance from the seed and has been found to become less complex with distance, which is potentially easier for the cell to repair. Close to the seed α-particles contribute the majority of dose, however the contribution from other particles cannot be neglected and may influence the choice of seed spacing.

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.

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.

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.

Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes.

Programmed DNA double-strand break (DSB) formation is a crucial feature of meiosis in most organisms. DSBs initiate recombination-mediated linking of homologous chromosomes, which enables correct chromosome segregation in meiosis. DSBs are generated on chromosome axes by heterooligomeric focal clusters of DSB-factors. Whereas DNA-driven protein condensation is thought to assemble the DSB-machinery, its targeting to chromosome axes is poorly understood. We uncover in mice that efficient biogenesis of DSB-machinery clusters requires seeding by axial IHO1 platforms. Both IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), suggesting that DDK contributes to the control of the axial DSB-machinery. Furthermore, we show that axial IHO1 platforms are based on an interaction between IHO1 and the chromosomal axis component HORMAD1. IHO1-HORMAD1-mediated seeding of the DSB-machinery on axes ensures sufficiency of DSBs for efficient pairing of homologous chromosomes. Without IHO1-HORMAD1 interaction, residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, recombination initiation is ensured by complementary pathways that differentially support seeding and growth of DSB-machinery clusters, thereby synergistically enabling DSB-machinery condensation on chromosomal axes.

Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination.

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.

TRIM33 loss in multiple myeloma is associated with genomic instability and sensitivity to PARP inhibitors.

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.

Sequential requirements for distinct Polθ domains during theta-mediated end joining.

DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.

Replication protein-A, RPA, plays a pivotal role in the maintenance of recombination checkpoint in yeast meiosis.

DNA double-strand breaks (DSBs) activate DNA damage responses (DDRs) in both mitotic and meiotic cells. A single-stranded DNA (ssDNA) binding protein, Replication protein-A (RPA) binds to the ssDNA formed at DSBs to activate ATR/Mec1 kinase for the response. Meiotic DSBs induce homologous recombination monitored by a meiotic DDR called the recombination checkpoint that blocks the pachytene exit in meiotic prophase I. In this study, we further characterized the essential role of RPA in the maintenance of the recombination checkpoint during Saccharomyces cerevisiae meiosis. The depletion of an RPA subunit, Rfa1, in a recombination-defective dmc1 mutant, fully alleviates the pachytene arrest with the persistent unrepaired DSBs. RPA depletion decreases the activity of a meiosis-specific CHK2 homolog, Mek1 kinase, which in turn activates the Ndt80 transcriptional regulator for pachytene exit. These support the idea that RPA is a sensor of ssDNAs for the activation of meiotic DDR. Rfa1 depletion also accelerates the prophase I delay in the zip1 mutant defective in both chromosome synapsis and the recombination, consistent with the notion that the accumulation of ssDNAs rather than defective synapsis triggers prophase I delay in the zip1 mutant.

MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

BACKGROUND: The repair of peripheral nerve injury poses a clinical challenge, necessitating further investigation into novel therapeutic approaches. In recent years, bone marrow mesenchymal stromal cell (MSC)-derived mitochondrial transfer has emerged as a promising therapy for cellular injury, with reported applications in central nerve injury. However, its potential therapeutic effect on peripheral nerve injury remains unclear. METHODS: We established a mouse sciatic nerve crush injury model. Mitochondria extracted from MSCs were intraneurally injected into the injured sciatic nerves. Axonal regeneration was observed through whole-mount nerve imaging. The dorsal root ganglions (DRGs) corresponding to the injured nerve were harvested to test the gene expression, reactive oxygen species (ROS) levels, as well as the degree and location of DNA double strand breaks (DSBs). RESULTS: The in vivo experiments showed that the mitochondrial injection therapy effectively promoted axon regeneration in injured sciatic nerves. Four days after injection of fluorescently labeled mitochondria into the injured nerves, fluorescently labeled mitochondria were detected in the corresponding DRGs. RNA-seq and qPCR results showed that the mitochondrial injection therapy enhanced the expression of Atf3 and other regeneration-associated genes in DRG neurons. Knocking down of Atf3 in DRGs by siRNA could diminish the therapeutic effect of mitochondrial injection. Subsequent experiments showed that mitochondrial injection therapy could increase the levels of ROS and DSBs in injury-associated DRG neurons, with this increase being correlated with Atf3 expression. ChIP and Co-IP experiments revealed an elevation of DSB levels within the transcription initiation region of the Atf3 gene following mitochondrial injection therapy, while also demonstrating a spatial proximity between mitochondria-induced DSBs and CTCF binding sites. CONCLUSION: These findings suggest that MSC-derived mitochondria injected into the injured nerves can be retrogradely transferred to DRG neuron somas via axoplasmic transport, and increase the DSBs at the transcription initiation regions of the Atf3 gene through ROS accumulation, which rapidly release the CTCF-mediated topological constraints on chromatin interactions. This process may enhance spatial interactions between the Atf3 promoter and enhancer, ultimately promoting Atf3 expression. The up-regulation of Atf3 induced by mitochondria further promotes the expression of downstream regeneration-associated genes and facilitates axon regeneration.

A Novel Interaction of Slug (SNAI2) and Nuclear Actin.

Actin is a protein of central importance to many cellular functions. Its localization and activity are regulated by interactions with a high number of actin-binding proteins. In a yeast two-hybrid (Y2H) screening system, snail family transcriptional repressor 2 (SNAI2 or slug) was identified as a yet unknown potential actin-binding protein. We validated this interaction using immunoprecipitation and analyzed the functional relation between slug and actin. Since both proteins have been reported to be involved in DNA double-strand break (DSB) repair, we focused on their interaction during this process after treatment with doxorubicin or UV irradiation. Confocal microscopy elicits that the overexpression of actin fused to an NLS stabilizes complexes of slug and γH2AX, an early marker of DNA damage repair.

LP-184, a Novel Acylfulvene Molecule, Exhibits Anticancer Activity against Diverse Solid Tumors with Homologous Recombination Deficiency.

Homologous recombination (HR)-related gene alterations are present in a significant subset of prostate, breast, ovarian, pancreatic, lung, and colon cancers rendering these tumors as potential responders to specific DNA damaging agents. A small molecule acylfulvene prodrug, LP-184, metabolizes to an active compound by the oxidoreductase activity of enzyme prostaglandin reductase 1 (PTGR1), which is frequently elevated in multiple solid tumor types. Prior work demonstrated that cancer cell lines deficient in a spectrum of DNA damage repair (DDR) pathway genes show increased susceptibility to LP-184. Here, we investigated the potential of LP-184 in targeting multiple tumors with impaired HR function and its mechanism of action as a DNA damaging agent. LP-184 induced elevated DNA double-strand breaks in HR deficient (HRD) cancer cells. Depletion of key HR components BRCA2 or ataxia telangiectasia mutated (ATM) in cancer cells conferred up to 12-fold increased sensitivity to the LP-184. LP-184 showed nanomolar potency in a diverse range of HRD cancer models, including prostate cancer organoids, leiomyosarcoma cell lines, and patient-derived tumor graft models of lung, pancreatic, and prostate cancers. LP-184 demonstrated complete, durable tumor regression in 10 patient-derived xenograft (PDX) models of HRD triple-negative breast cancer (TNBC) including those resistant to PARP inhibitors (PARPi). LP-184 further displayed strong synergy with PARPi in ovarian and prostate cancer cell lines as well as in TNBC PDX models. These preclinical findings illustrate the potential of LP-184 as a pan-HRD cancer therapeutic. Taken together, our results support continued clinical evaluation of LP-184 in a large subset of HRD solid tumors. SIGNIFICANCE: New agents with activity against DDR-deficient solid tumors refractory to standard-of-care therapies are needed. We report multiple findings supporting the potential for LP-184, a novel alkylating agent with three FDA orphan drug designations, to fill this void clinically: strong nanomolar potency; sustained, durable regression of solid tumor xenografts; synthetic lethality with HR defects. LP-184 adult phase IA trial to assess safety in advanced solid tumors is ongoing.

How a Single 5 eV Electron Can Induce Double-Strand Breaks in DNA: A Time-Dependent Density Functional Theory Study.

Low-energy (<20 eV) electrons (LEEs) can resonantly interact with DNA to form transient anions (TAs) of fundamental units, inducing single-strand breaks (SSBs), and cluster damage, such as double-strand breaks (DSBs). Shape resonances, which arise from electron capture in a previously unfilled orbital, can induce only a SSB, whereas a single core-excited resonance (i.e., two electrons in excited orbitals of the field of a hole) has been shown experimentally to cause cluster lesions. Herein, we show from time-dependent density functional theory (TDDFT) that a core-excited resonance can produce a DSB, i.e., a single 5 eV electron can induce two close lesions in DNA. We considered the nucleotide with the G-C base pair (ds[5'-G-3']) as a model for electron localization in the DNA double helix and calculated the potential energy surfaces (PESs) of excited states of the ground-state TA of ds[5'-G-3'], which correspond to shape and core-excited resonances. The calculations show that shape TAs start at ca. 1 eV, while core-excited TAs occur only above 4 eV. The energy profile of each excited state and the corresponding PES are obtained by simultaneously stretching both C5'-O5' bonds of ds[5'-G-3']. From the nature of the PES, we find two dissociative (σ*) states localized on the PO4 groups at the C5' sites of ds[5'-G-3']. The first σ* state at 1 eV is due to a shape resonance, while the second σ* state is induced by a core-excited resonance at 5.4 eV. As the bond of the latter state stretches and arrives close to the dissociation limit, the added electron on C transfers to C5' phosphate, thus demonstrating the possibility of producing a DSB with only one electron of ca. 5 eV.

Estimation of the relative biological effectiveness for double strand break induction of clinical kilovoltage beams using Monte Carlo simulations.

BACKGROUND: The Relative Biological Effectiveness (RBE) of kilovoltage photon beams has been previously investigated in vitro and in silico using analytical methods. The estimated values range from 1.03 to 1.82 depending on the methodology and beam energies examined. PURPOSE: The focus of this work was to independently estimate RBE values for a range of clinically used kilovoltage beams (70-200 kVp) while investigating the suitability of using TOPAS-nBio for this task. METHODS: Previously validated spectra of clinical beams were used to generate secondary electron spectra at several depths in a water tank phantom via TOPAS Monte Carlo (MC) simulations. Cell geometry was irradiated with the secondary electrons in TOPAS-nBio MC simulations. The deposited dose and the calculated number of DNA strand breaks were used to estimate RBE values. RESULTS: Monoenergetic secondary electron simulations revealed the highest direct and indirect double strand break yield at approximately 20 keV. The average RBE value for the kilovoltage beams was calculated to be 1.14. CONCLUSIONS: TOPAS-nBio was successfully used to estimate the RBE values for a range of clinical radiotherapy beams. The calculated value was in agreement with previous estimates, providing confidence in its clinical use in the future.