DNA Double-strand Break Repair Research Articles

From Zebrafish To Humans: In Silico Comparative Study of RAD50 Sequences

DNA damage, particularly the occurrence of DNA double-strand breaks (DSBs), presents a significant hazard to the integrity and viability of cells. Improper repair of DSBs can result in chromosomal alterations, oncogenic changes, or cell demise. The MRE11-RAD50-NBS1 (MRN) complex plays a crucial role in DNA repair and signaling under the Ataxia Telangiectasia Mutated (ATM) kinase regulation. In this study, we employed comprehensive computational techniques to analyze the structure of RAD50 in Danio rerio (Zebrafish), utilized as a model organism. Additionally, we conducted in silico assessments of RAD50 from both Zebrafish and humans, comparing their characteristics. The substantial sequence resemblance between DrRAD50 and HsRAD50 suggests that DrRAD50 could potentially serve as a valuable model for HsRAD50. However, it is important to acknowledge that sequence similarity alone does not necessarily imply functional equivalence. Further functional studies are needed to confirm the extent of their functional similarities. By examining the secondary and tertiary protein structures of RAD50, we observed a notable likeness between Zebrafish and Human RAD50 proteins. In silico analysis demonstrated that the sequence of RAD50 in zebrafish shares 70% similarity with the human RAD50 protein.

RAD50 Deficiency and Its Effects on Zebrafish Embryonic Development and DNA Repair Mechanisms

The MRE11-RAD50-NBS1 (MRN) complex is essential in detecting, signaling, and repairing DNA double-strand breaks (DSBs), thus maintaining genomic integrity. Mutations in RAD50 are linked to severe conditions such as microcephaly, mental retardation, and growth retardation in humans. This study investigates the developmental impact of RAD50 protein disruption in zebrafish embryos. Zebrafish embryos were treated with MIRIN (35 µM) to inhibit RAD50 and subsequently exposed to gamma-ray irradiation (15 Gy) to analyze the role of RAD50 in managing DNA damage during embryogenesis. Time-point analysis indicated that inhibiting RAD50 and ATM proteins during early embryonic stages (at 1 hpf) leads to increased embryonic mortality and abnormalities. These adverse effects were exacerbated by irradiation, underscoring the critical role of RAD50 in DNA DSB repair. The study concludes that RAD50 deficiencies can lead to embryonic lethality and human deformities due to the inability of tissues to repair DNA DSBs effectively.

Non-classical action of Ku70 promotes Treg suppressive function through a FOXP3-dependent mechanism in lung adenocarcinoma.

Ku70, a DNA repair protein, binds to the damaged DNA ends and orchestrates the recruitment of other proteins to facilitate repair of DNA double-strand breaks. Besides its essential role in DNA repair, several studies have highlighted non-classical functions of Ku70 in cellular processes. However, its function in immune homeostasis and anti-tumor immunity remains unknown. Here, we discovered a marked association between elevated Ku70 expression and unfavorable prognosis in lung adenocarcinoma, focusing specifically on increased Ku70 levels in tumor-infiltrated Treg cells. Using a lung-colonizing tumor model of in mice with Treg-specific Ku70 deficiency, we demonstrated that deletion of Ku70 in Treg cells led to a stronger anti-tumor response and slower tumor growth due to impaired immune-suppressive capacity of Treg cells. Furthermore, we confirmed that Ku70 played a critical role in sustaining the suppressive function of human Treg cells. We found that Ku70 bound to FOXP3 and occupied FOXP3-bound genomic sites to support its transcriptional activities. These findings not only unveil a non-homologous end joining (NHEJ)-independent role of Ku70 crucial for Treg suppressive function, but also underscore the potential of targeting Ku70 as an effective strategy in cancer therapy, aiming to both restrain cancer cells and enhance pulmonary anti-tumor immunity.

Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs)and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin(SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids andefficient DNA repair.

NBS1 dePARylation by NUDT16 is critical for DNA double-strand break repair.

NBS1, a protein linked to the autosomal recessive disorder Nijmegen breakage syndrome, plays an essential role in the DNA damage response and DNA repair. Despite its importance, the mechanisms regulating NBS1 and the impact of this regulation on DNA repair processes remain obscure. In this study, we discovered a new post-translational modification of NBS1, ADP-ribosylation. This modification can be removed by the NUDT16 hydrolase. The loss of NUDT16 results in a reduction of NBS1 protein levels due to NBS1 PARylation-dependent ubiquitination and degradation, which is mediated by the PAR-binding E3 ubiquitin ligase, RNF146. Importantly, ADP-ribosylation of NBS1 is crucial for its localization at DSBs and its involvement in homologous recombination (HR) repair. Additionally, the NUDT16-NBS1 interaction is regulated in response to DNA damage, providing further rationale for NBS1 regulation by NUDT16 hydrolase. In summary, our study unveils the critical role of NUDT16 in governing both the stability of NBS1 and recruitment of NBS1 to DNA double-strand breaks, providing novel insights into the regulation of NBS1 in the HR repair pathway.

NF-κB in Alzheimer’s Disease: Friend or Foe? Opposite Functions in Neurons and Glial Cells

Alzheimer’s disease (AD) is a devasting neurodegenerative disease afflicting mainly glutamatergic neurons together with a massive neuroinflammation mediated by the transcription factor NF-κB. A 65%-plus increase in Alzheimer’s patients by 2050 might be a major threat to society. Hallmarks of AD are neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau and amyloid beta (Aβ) plaques. Here, we review the potential involvement of transcription factor NF-κB by hereditary mutations of the tumor necrosis factor pathway in AD patients. One of the greatest genetic risk factors is APOE4. Recently, it was shown that the APOE4 allele functions as a null allele in human astrocytes not repressing NF-κB anymore. Moreover, NF-κB seems to be involved in the repair of DNA double-strand breaks during healthy learning and memory, a function blunted in AD. NF-κB could be a friend to healthy neurons by repressing apoptosis and necroptosis. But a loss of neuronal NF-κB and activation of glial NF-κB in AD makes it a foe of neuronal survival. Hopeful therapies include TNFR2 receptor bodies relieving the activation of glial NF-κB by TNFα.

Role of TRIP13 in human cancer development.

As an AAA + ATPase, thyroid hormone receptor interacting protein 13 (TRIP13) primarily functions in DNA double-strand break repair, chromosome recombination, and cell cycle checkpoint regulation; aberrant expression of TRIP13 can result in chromosomal instability (CIN). According to recent research, TRIP13 is aberrantly expressed in a variety of cancers, and a patient's poor prognosis and tumor stage are strongly correlated with high expression of TRIP13. Tumor cell and subcutaneous xenograft growth can be markedly inhibited by TRIP13 knockdown or TRIP13 inhibitor administration. In the initiation and advancement of human malignancies, TRIP13 seems to function as an oncogene. Based on available data, TRIP13 may function as a biological target and biomarker for cancer. The creation of inhibitors that specifically target TRIP13 may present novel approaches to treating cancer.

Ribosomal S6 kinase (RSK) plays a critical role in DNA damage response via the phosphorylation of histone lysine demethylase KDM4B

BackgroundEpigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.MethodsKDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.ResultsRSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.ConclusionsOverall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.

Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair

DNA double-strand breaks (DSBs) must be properly repaired within diverse chromatin domains to maintain genome stability. Whereas euchromatin has an open structure and is associated with transcription, facultative heterochromatin is essential to silence developmental genes and forms compact nuclear condensates, called polycomb bodies. Whether the specific chromatin properties of facultative heterochromatin require distinct DSB repair mechanisms remains unknown. Here, we integrate single DSB systems in euchromatin and facultative heterochromatin in Drosophila melanogaster and find that heterochromatic DSBs rapidly move outside polycomb bodies. These DSB movements coincide with a break-proximal reduction in the canonical heterochromatin mark histone H3 Lysine 27 trimethylation (H3K27me3). We demonstrate that DSB movement and loss of H3K27me3 at heterochromatic DSBs depend on the histone demethylase dUtx. Moreover, loss of dUtx specifically disrupts completion of homologous recombination at heterochromatic DSBs. We conclude that DSBs in facultative heterochromatin require dUtx-mediated loss of H3K27me3 to promote DSB movement and repair.

The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

RPS19 and RPL5, the most commonly mutated genes in Diamond Blackfan anemia, impact DNA double-strand break repair.

Diamond Blackfan anemia (DBA) is caused by germline heterozygous loss-of-function pathogenic variants (PVs) in ribosomal protein (RP) genes, most commonly RPS19 and RPL5 . In addition to red cell aplasia, individuals with DBA are at increased risk of various cancers. Importantly, the mechanism(s) underlying cancer predisposition are poorly understood. We found that DBA patient-derived lymphoblastoid cells had persistent γ-H2AX foci following ionizing radiation (IR) treatment, suggesting DNA double-strand break (DSB) repair defects. RPS19- and RPL5-knocked down (KD) CD34+ cells had delayed repair of IR-induced DSBs, further implicating these RPs in DSB repair. Assessing the impact of RPS19- and RPL5-KD on specific DSB repair pathways, we found RPS19-KD decreased the efficiency of pathways requiring extensive end-resection, whereas RPL5-KD increased end-joining pathways. Additionally, RAD51 was reduced in RPS19- and RPL5-KD and RPS19- and RPL5-mutated DBA cells, whereas RPS19-deficient cells also had a reduction in PARP1 and BRCA2 proteins. RPS19-KD cells had an increase in nuclear RPA2 and a decrease in nuclear RAD51 foci post-IR, reflective of alterations in early, critical steps of homologous recombination. Notably, RPS19 and RPL5 interacted with poly(ADP)-ribose chains noncovalently, were recruited to DSBs in a poly(ADP)-ribose polymerase activity-dependent manner, and interacted with Ku70 and histone H2A. RPL5's recruitment, but not RPS19's, also required p53, suggesting that RPS19 and RPL5 directly participate in DSB repair via different pathways. We propose that defective DSB repair arising from haploinsufficiency of these RPs may underline the cancer predisposition in DBA.

GLTSCR1 deficiency promotes colorectal cancer development through regulating non-homologous end joining.

Non-homologous end joining (NHEJ), as one major pathway of DNA double-strand break (DSB) repair, could cause genomic instability, which plays pivotal roles in cancer development. While, chromatin remodeling complexes dictate the selection and orchestration of DSB repair pathways by regulating chromatin dynamics. However, the crosstalk between NHEJ and chromatin remodeling in cancer progress remains unclear. In this study, deficiency of GLTSCR1 causes resistance to DNA damage in colorectal cancer (CRC) cells by promoting NHEJ repair efficiency. Mechanistically, GLTSCR1 interacts with BRD9 to engage in the assembly of the non-canonical BAF complex (GBAF). However, GLTSCR1 deficiency disrupts GBAF and triggers the ubiquitination degradation of BRD9. Furthermore, GLTSCR1 deficiency causes aberrant opening in the promoter region of NHEJ repair-associated genes, which promotes CRC development. While, GLTSCR1 and its binding partner BRD9 are not directly involved in assembling NHEJ repair machinery; instead, they regulate the DNA accessibility of NHEJ repair-associated genes. Collectively, our findings confirm GLTSCR1 deficiency as a critical regulatory event of the NHEJ pathway in CRC development, which might require different therapeutic strategy for GLTSCR1 wild-type and mutant CRC.

Isolation and purification of DNA double-strand break repair intermediates for understanding complex molecular mechanisms.

Branched DNA molecules are key intermediates in the molecular pathways of DNA replication, repair and recombination. Understanding their structural details, therefore, helps to envisage the mechanisms underlying these processes. While the configurations of DNA molecules can be effectively analysed in bulk using gel electrophoresis techniques, direct visualization provides a complementary single-molecule approach to investigating branched DNA structures. However, for microscopic examination, the sample needs to be free from impurities that could obscure the molecules of interest, and free from the bulk of unwanted non-specific DNA molecules that would otherwise dominate the field of view. Additionally, in the case of recombination intermediates, the length of the DNA molecules becomes an important factor to consider since the structures can be spread over a large distance on the chromosome in vivo. As a result, apart from sample purity, efficient isolation of large-sized DNA fragments without damaging their branched structures is crucial for further analysis. These factors are illustrated by the example of DNA double-strand break repair in the bacterium E. coli. In E. coli recombination intermediates may be spread over a distance of 40 kb which constitutes less than 1% of the 4.6 Mb genome. This study reveals ways to overcome some of the technical challenges that are associated with the isolation and purification of large and complex branched DNA structures using E. coli DNA double-strand break repair intermediates. High-molecular weight and branched DNA molecules do not run into agarose gels subjected to electrophoresis. However, they can be extracted from the wells of the gels if they are agarose embedded, by using β-agarase digestion, filtration, and concentration. Furthermore, a second round of gel electrophoresis followed by purification is recommended to enhance the purity of the specific DNA samples. These preliminary findings may prove to be pioneering for various single-molecule analyses of large and complex DNA molecules of DNA replication, repair and recombination.

VGLL3 modulates chemosensitivity through promoting DNA double-strand break repair.

Transcription cofactor vestigial-like 3 (VGLL3), as a master regulator of female-biased autoimmunity, also functions in tumor development, while the underlying mechanisms remain largely elusive. Here, we report that VGLL3 plays an important role in DNA damage response (DDR). VGLL3 can be recruited to damage sites in a PARylation-dependent manner. VGLL3 depletion impairs the accumulation of RNF8 and RAD51 at sites of DNA damage, leading to reduced homologous recombination efficiency and increased cellular sensitivity to chemotherapeutic drugs. Mechanistically, VGLL3 can prevent CtIP from KLHL15-mediated ubiquitination and degradation through competitive binding with KLHL15 and, meanwhile, stabilize MDC1 by limiting TRIP12-MDC1 but promoting USP7-MDC1 associations for optimal RNF8 signaling initiation. Consistently, VGLL3 depletion delays tumor development and sensitizes the xenografts to etoposide treatment. Overall, our results reveal an unexpected role of VGLL3 in DDR, which is distinct from its transcriptional cofactor function and not conserved among VGLL family members.

Effects of the auxin-dependent degradation of the cohesin and condensin complexes on the repair of distant DNA double-strand breaks in mouse embryonic stem cells.

The SMC protein family, including cohesin and condensin I/II, plays a pivotal role in maintaining the topological structure of chromosomes and influences many cellular processes, notably the repair of double-stranded DNA breaks (DSBs). The cohesin complex impacts DSB repair by spreading γH2AX signal and containing DNA ends in close proximity by loop extrusion. Cohesin supports DNA stability by sister chromatid cohesion during the S/G2 phase, which limits DNA end mobility. Cohesin knockdown was recently shown to stimulate frequencies of genomic deletions produced by distant paired DSBs, but does not affect DNA repair of a single or close DSBs. We examined how auxin-inducible protein degradation of Rad21 (cohesin) or Smc2 (condensins I+II) changes the frequencies of rearrangements between paired distant DSBs in mouse embryonic stem cells (mESCs). We used Cas9 RNP nucleofection to generate deletions and inversions with high efficiency without additional selection. We determined optimal Neon settings and deletion appearance timings. Two strategies for auxin addition were tested (4 independent experiments in total). We examined deletion/inversion frequencies for two regions spanning 3.5 and 3.9 kbp in size. Contrary to expectations, in our setting, Rad21 depletion did not increase deletion/inversion frequencies, not even for the region with an active Ctcf boundary. We actually observed a 12 % decrease in deletions (but not inversions). At the same time, double condensin depletion (Smc2 degron line) demonstrated high biological variability between experiments, complicating the analysis, and requires additional examination in the future. TIDE analysis revealed that editing frequency was consistent (30-50 %) for most experiments with a minor decrease after auxin addition. In the end, we discuss the Neon/ddPCR method for deletion generation and detection in mESCs.

A Site-Specific Click Chemistry Approach to Di-ubiquitylate H1 Variants Reveals Position-dependent Stimulation of the DNA Repair Protein RNF168.

Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DNA double-strand break (DSB) repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, direct experimental evidence of this mechanism has been elusive, primarily due to the lack of methods to specifically poly-ubiquitylate H1. Here, we developed a versatile click-chemistry approach to covalently link multiple proteins in a site-specific, controlled, and stepwise manner. Applying this method, we synthesized H1 constructs bearing triazole-linked di-ubiquitin on four DNA repair-associated ubiquitylation hotspots (H1KxUb2, at K17, 46, 64 and 96). Integrated into nucleosome arrays, the H1KxUb2 variants stimulated H2A ubiquitylation by RNF168 in a position-dependent manner, with H1K17Ub2 showing the strongest RNF168 activation effect. Moreover, we show that di-ubiquitin binding is the driving force underlying RNF168 recruitment, introducing H1K17Ub2 into living U-2 OS cells. Together, our results support the hypothesis of poly-ubiquitylated H1 guiding RNF168 recruitment to DSB sites. Moreover, we demonstrate how the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers.

Quantitation of the DNA-dependent protein kinase inhibitor peposertib (M3814) and metabolite in human plasma by LC-MS/MS.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4μm, 2× 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

RPS15 Coordinates with CtIP to Facilitate Homologous Recombination and Confer Therapeutic Resistance in Breast Cancer.

The repair of DNA double-strand breaks (DSBs) through homologous recombination (HR) is vital for maintaining the stability and integrity of the genome. RNA binding proteins (RBPs) intricately regulate the DNA damage repair process, yet the precise molecular mechanisms underlying their function remain incompletely understood. In this study, we highlight the pivotal role of RPS15, a representative RBP, in homologous recombination repair. Specifically, we demonstrate that RPS15 promotes DNA end resection, a crucial step in homologous recombination. Notably, we identify an interaction between RPS15 and CtIP, a key factor in homologous recombination repair. This interaction is essential for CtIP recruitment to DSB sites, subsequent RPA coating, and RAD51 replacement, all critical steps in efficient homologous recombination repair and conferring resistance to genotoxic treatments. Functionally, suppressing RPS15 expression sensitizes cancer cells to X-ray radiation and enhances the therapeutic synergistic effect of PARP1 inhibitors in breast cancer cells. In summary, our findings reveal that RPS15 promotes DNA end resection to ensure effective homologous recombination repair, suggesting its potential as a therapeutic target in cancer treatment.

53BP1 regulates the self-renewal ability of neural stem/progenitor cells through modulating mitochondrial homeostasis

The regulation of intracellular reactive oxygen species (ROS) levels is important for maintaining the self-renewal ability of neural stem/progenitor cells (NSCs). In this study, we demonstrate that 53BP1, a DNA damage response factor known to facilitate the repair of DNA double-strand breaks, supports the maintenance of NSC stemness. ReNcell VM human NSCs with depleted 53BP1 exhibited reduced self-renewal ability compared with control NSCs, as revealed by a decrease in neurosphere size and an increase in differentiation into neural or glial cells within an NSC culture. Furthermore, 53BP1 depletion elevated cellular ROS levels, accompanied by mitochondrial abnormalities. The reduced self-renewal ability and elevated ROS levels in 53BP1-deficient NSCs were restored with the treatment of a radical scavenger, N-acetyl-l-cysteine. In addition, we investigated the functional relationship in the NSC self-renewal ability between 53BP1 and ataxia-telangiectasia mutated (ATM) or forkhead box O3a (FOXO3a), factors required for mitochondrial homeostasis, and the maintenance of NSC stemness. We found that ATM inhibition or FOXO3a deficiency, in addition to 53BP1 deficiency, did not induce further NSC stemness impairment. Collectively, our findings show that 53BP1, by cooperatively functioning with ATM and FOXO3a, supports the maintenance of NSC stemness by modulating mitochondrial homeostasis.

Microhomology-Mediated End-Joining Chronicles: Tracing the Evolutionary Footprints of Genome Protection.

The fidelity of genetic information is essential for cellular function and viability. DNA double-strand breaks (DSBs) pose a significant threat to genome integrity, necessitating efficient repair mechanisms. While the predominant repair strategies are usually accurate, paradoxically, error-prone pathways also exist. This review explores recent advances and our understanding of microhomology-mediated end joining (MMEJ), an intrinsically mutagenic DSB repair pathway conserved across organisms. Central to MMEJ is the activity of DNA polymerase theta (Polθ), a specialized polymerase that fuels MMEJ mutagenicity. We examine the molecular intricacies underlying MMEJ activity and discuss its function during mitosis, where the activity of Polθ emerges as a last-ditch effort to resolve persistent DSBs, especially when homologous recombination is compromised. We explore the promising therapeutic applications of targeting Polθ in cancer treatment and genome editing. Lastly, we discuss the evolutionary consequences of MMEJ, highlighting its delicate balance between protecting genome integrity and driving genomic diversity.