Search

Genetic mutations significantly contribute to the onset of diseases, with over half of the cases caused by single-nucleotide mutations. Advances in gene editing technologies have enabled precise editing and correction of mutated genes, offering effective treatment methods for genetic disorders. CRISPR/Cas9, despite its power, poses risks of inducing gene mutations due to DNA double-strand breaks (DSB). The advent of base editing (BE) and prime editing (PE) has mitigated these risks by eliminating the hazards associated with DNA DSBs, allowing for more precise gene editing. This breakthrough lays a solid foundation for the clinical application of gene editing technologies. This review discusses the principles, development, and applications of PE gene editing technology in various genetic mutation-induced diseases., (© 2024 Federation of American Societies for Experimental Biology.)

This study investigated the induction of DNA double-strand breaks (DSBs) in the hTERT-immortalized normal human diploid epithelial cells (RPE1-hTERT) continuously exposed to 6000 Bq/ml of tritiated water (HTO) and organically bound tritium (OBT). The relationship of the DSBs induction with the intracellular amount as well as the localization of tritium was also examined. Tritium-labeled thymidine (3H-Thy) and palmitic acid (3H-PA) were used as OBT. The average number of DSBs, which were indicated as co-localized foci of 53BP1 with phosphorylated H2AX, per cell was higher in the order of treatments with 3H-Thy, 3H-PA, and HTO. This order was consistent with that of tritium localization in the insoluble nuclear fraction but not with the intracellular amount of tritium. In addition to the intracellular amount of tritium, we showed that the subcellular localization of tritium is an important factor in cellular effects stimulated by DSBs., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We established two types of site-specific DNA double strand breaks (DSB) induction systems to elucidate factors which affect the efficiency or quality of DSB repair. For mutation assays, a site specific DSB was generated by the transient expression of a zinc finger nuclease which targets the human HPRT1 gene. A cell line in which time and site specific DSBs can be generated in a HR reporter construct was used for homology-directed repair (HR) analysis. By using these two systems, we investigated the effects of PI3-kinase inhibitors on the efficiency and quality of DSB repair. Ataxia telangiectasia mutated (ATM) kinase inhibition resulted a decrease in mutant frequency and a slight increase in deletion-type mutations accompanied by microhomology. Furthermore, the HR frequency increased significantly when ATM kinase activity was inhibited. Thus, ATM kinase activity might be involved in the suppression of DSB end resection, and this may promote DSB repair through canonical non-homologous end joining., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

O 6 -methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme. The roles of this enzyme in immune cells remain unclear. In this study, we explored the roles of MGMT in bone marrow-derived murine macrophages (BMMs) via the use of MGMT knockout (KO) mice. Loss of MGMT altered the response to TLR3 agonists (poly (I:C)), such as dampening the production of TNFα and IL-6. Increased DNA double-strand breaks (DSBs) were observed in MGMT-KO macrophages but did not result in increased cell death. MGMT localized to both nuclei and mitochondria at increasing levels during poly (I:C) stimulation. MGMT deficiency increased the production of mitochondrial reactive oxygen species (mtROS), which was correlated with increased mitophagy. The underlying mechanism involves mediation through activation of the AMPKα pathway. Taken together, our findings reveal the roles of MGMT in macrophages in regulating the response to TLR3, which links DSBs to mtROS and mitophagy via the AMPKα pathway. These roles may have consequences for the inflammatory response and chronic inflammation., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Replication-dependent DNA double-strand breaks are harmful lesions preferentially repaired by homologous recombination (HR), a process that requires processing of DNA ends to allow RAD51-mediated strand invasion. End resection and subsequent repair are two intertwined processes, but the mechanism underlying their execution is still poorly appreciated. The WRN helicase is one of the crucial factors for end resection and is instrumental in selecting the proper repair pathway. Here, we reveal that ordered phosphorylation of WRN by the CDK1, ATM and ATR kinases defines a complex regulatory layer essential for correct long-range end resection, connecting it to repair by HR. We establish that long-range end resection requires an ATM-dependent phosphorylation of WRN at Ser1058 and that phosphorylation at Ser1141, together with dephosphorylation at the CDK1 site Ser1133, is needed for the proper metabolism of RAD51 foci and RAD51-dependent repair. Collectively, our findings suggest that regulation of WRN by multiple kinases functions as a molecular switch to allow timely execution of end resection and repair at replication-dependent DNA double-strand breaks., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To ensure the integrity of our genetic code, a coordinated network of signalling and repair proteins, known as the DNA damage response (DDR), detects and repairs DNA insults, the most toxic being double-strand breaks (DSBs). Tudor interacting repair regulator (TIRR) is a key factor in DSB repair, acting through its interaction with p53 binding protein 1 (53BP1). TIRR is also an RNA binding protein, yet its role in RNA regulation during the DDR remains elusive. Here, we show that TIRR selectively binds to a subset of messenger RNAs (mRNAs) in response to DNA damage. Upon DNA damage, TIRR interacts with the nuclear export protein Exportin-1 through a nuclear export signal. Furthermore, TIRR plays a crucial role in the modulation of RNA processing bodies (PBs). TIRR itself and TIRR-bound RNA co-localize with PBs, and TIRR depletion results in nuclear RNA retention and impaired PB formation. We also suggest a potential link between TIRR-regulated RNA export and efficient DDR. This work reveals intricate involvement of TIRR in orchestrating mRNA nuclear export and storage within PBs, emphasizing its significance in the regulation of RNA-mediated DDR., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Understanding the intricacies of homologous recombination during meiosis is crucial for reproductive biology. However, the role of alternative splicing (AS) in DNA double-strand breaks (DSBs) repair and synapsis remains elusive. In this study, we investigated the impact of conditional knockout (cKO) of the splicing factor gene Bcas2 in mouse germ cells, revealing impaired DSBs repair and synapsis, resulting in non-obstructive azoospermia (NOA). Employing crosslinking immunoprecipitation and sequencing (CLIP-seq), we globally mapped BCAS2 binding sites in the testis, uncovering its predominant association with 5' splice sites (5'SS) of introns and a preference for GA-rich regions. Notably, BCAS2 exhibited direct binding and regulatory influence on Trp53bp1 (codes for 53BP1) and Six6os1 through AS, unveiling novel insights into DSBs repair and synapsis during meiotic prophase I. Furthermore, the interaction between BCAS2, hnRNPH1, and SRSF3 was discovered to orchestrate Trp53bp1 expression via AS, underscoring its role in meiotic prophase I DSBs repair. In summary, our findings delineate the indispensable role of BCAS2-mediated post-transcriptional regulation in DSBs repair and synapsis during male meiosis. This study provides a comprehensive framework for unraveling the molecular mechanisms governing the post-transcriptional network in male meiosis, contributing to the broader understanding of reproductive biology., (© 2024. The Author(s).)

Efficient DNA double strand break (DSB) repair is necessary for genomic stability and determines efficacy of DNA damaging cancer therapeutics. Spatiotemporal dynamics and post-translational modifications of repair proteins at DSBs dictate repair efficacy. Here, we identified a non-canonical function of GCN5 in regulating both HR and NHEJ repair post genotoxic stress. Mechanistically, genotoxic stress induced GCN5 recruitment to DSBs. GCN5 PARylation by PARP1 was essential for its recruitment, acetyltransferase activity and DSB repair function. Liquid chromatography-mass spectrometry (LC-MS) identified DNA-PKcs as part of GCN5 interactome. In-vitro acetyltransferase assays revealed that GCN5 acetylates DNA-PKcs at K3241 residue, a prerequisite for DNA-PKcs S2056 phosphorylation and DSB recruitment. Alongside, ChIP-qPCR revealed GCN5 mediates transcription of PRKDC via H3K27Ac acetylation in its promoter region (- 710 to - 554). Genetic perturbation of GCN5 also decreased CHEK1, NBN1, TP53BP1, POL-L transcription and abrogated ATM, BRCA1 activation. Accordingly, GCN5 loss led to persistent ɣ-H2AX foci formation, compromised in-vivo HR-NHEJ and caused GBM radio-sensitization. Importantly, PARP1 inhibition phenocopied GCN5 loss. Together, this study identifies an untraversed DSB repair function of GCN5 and provides mechanistic insights into transcriptional as well as post-translational regulation of pivotal HR-NHEJ factors. Alongside, it highlights the translational importance of PARP1-GCN5 axis in mediating GBM radio-resistance., (© 2024. The Author(s).)

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., Competing Interests: Conflicts of interest No authors have any conflicts of interest to declare., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

We previously developed an adeno-associated virus (AAV) Cas9 gene therapy for Angelman syndrome that integrated into the genome and prematurely terminated Ube3a-ATS. Here, we assessed the performance of 3 additional AAV vectors containing S. aureus Cas9 in vitro and in vivo, and 25 vectors containing N. meningitidis Cas9 in vitro, all targeting single sites within Ube3a-ATS. We found that none of these single-target gRNA vectors were as effective as multi-target gRNA vectors at reducing Ube3a-ATS expression in neurons. We also developed an anchored multiplex PCR sequencing method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including AAV integration and unresolved double-strand breaks. We found that integration of AAV was the most frequent editing event (67%-89% of all edits) at three different single target sites, surpassing insertions and deletions (indels). None of the most frequently observed indels were capable of blocking transcription when incorporated into a Ube3a-ATS minigene reporter, whereas two vector derived elements-the poly(A) and reverse promoter-reduced downstream transcription by up to 50%. Our findings suggest that the probability that a gene trapping AAV integration event occurs is influenced by which vector-derived element(s) are integrated and by the number of target sites., Competing Interests: Declaration of interests M.J.Z. previously served as a consultant to Asklepios BioPharmaceutical, Inc. M.J.Z., J.M.W., and H.M. are co-inventors on pending US Patent Application No. 16/976,386 entitled: Methods and compositions for treating Angelman syndrome. H.O.B. and J.K.N. declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Unlike mild DNA damage exposure, DNA damage repair (DDR) is reported to be ineffective in full-grown mammalian oocytes exposed to moderate or severe DNA damage. The underlying mechanisms of this weakened DDR are unknown. Here, we show that moderate DNA damage in full-grown oocytes leads to aneuploidy. Our data reveal that DNA-damaged oocytes have an altered, closed, chromatin state, and suggest that the failure to repair damaged DNA could be due to the inability of DDR proteins to access damaged loci. Our data also demonstrate that, unlike somatic cells, mouse and porcine oocytes fail to activate autophagy in response to DNA double-strand break-inducing treatment, which we suggest may be the cause of the altered chromatin conformation and inefficient DDR. Importantly, autophagy activity is further reduced in maternally aged oocytes (which harbor severe DNA damage), and its induction is correlated with reduced DNA damage in maternally aged oocytes. Our findings provide evidence that reduced autophagy activation contributes to weakened DDR in oocytes, especially in those from aged females, offering new possibilities to improve assisted reproductive therapy in women with compromised oocyte quality., (© 2024. The Author(s).)

Breast Cancer Type 1 Susceptibility Protein (BRCA)-1 existing in several functionally distinct complexes, promotes DNA repair of DNA double-strand breaks (DSBs). A recent study by Tang and colleagues identifies the lysine methyltransferase Disruptor of Telomeric Silencing 1-Like (DOT1L) involved in modifying Receptor-Associated Protein 80 (RAP80) to promote BRCA1-A complex localization and repair functions at DNA breaks. This study illuminates a potential therapeutic target for cancer radiotherapy., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The intracellular responses to DNA double-strand breaks (DSB) repair are crucial for genomic stability and play an essential role in cancer resistance. In addition to canonical DSB repair proteins, long non-coding RNAs (lncRNAs) have been found to be involved in this sophisticated network. In the present study, we performed a loss-of-function screen for a customized siRNA Premix Library to identify lncRNAs that participate in homologous recombination (HR) process. Among the candidates, we identified LINC01664 as a novel lncRNA required for HR repair. Furthermore, LINC01664 knockdown significantly increased the sensitivity of cancer cells to DNA damage agents such as ionizing radiation and genotoxic drugs. Mechanistically, LINC01664 interacted with Sirt1 promoter and then activated Sirt1 transcription, which contributed to HR-mediated DNA damage repair. In summary, our findings revealed a new mechanism of LINC01664 in DNA damage repair, providing evidence for a potential therapeutic strategy for eliminating the treatment bottlenecks caused by cancer resistance to chemotherapy and radiotherapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

Chloroplasts are organelles that are derived from a photosynthetic bacterium and have their own genome. Genome editing is a recently developing technology that allows for specific modifications of target sequences. The first successful application of genome editing in chloroplasts was reported in 2021, and since then, this research field has been expanding. Although the chloroplast genome of several dicot species can be stably modified by a conventional method, which involves inserting foreign DNAs into the chloroplast genome via homologous recombination, genome editing offers several advantages over this method. In this review, we introduce genome editing methods targeting the chloroplast genome and describe their advantages and limitations. So far, CRISPR/Cas systems are inapplicable for editing the chloroplast genome because guide RNAs, unlike proteins, cannot be efficiently delivered into chloroplasts. Therefore, protein-based enzymes are used to edit the chloroplast genome. These enzymes contain a chloroplast-transit peptide, the DNA-binding domain of transcription activator-like effector nuclease (TALEN), or a catalytic domain that induces DNA modifications. To date, genome editing methods can cause DNA double-strand break or introduce C:G-to-T:A and A:T-to-G:C base edits at or near the target sequence. These methods are expected to contribute to basic research on the chloroplast genome in many species and to be fundamental methods of plant breeding utilizing the chloroplast genome., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Histone modifications are catalyzed and recognized by specific proteins to regulate dynamic DNA metabolism processes. NSD2 is a histone H3 lysine 36 (H3K36)-specific methyltransferase that is associated with both various transcription regulators and DNA repair factors. Specifically, it has been implicated in the repair of DNA double-strand breaks (DSBs); however, the role of NSD2 during DSB repair remains enigmatic. Here, we show that NSD2 does not accumulate at DSB sites and that it is not further mobilized by DSB formation. Using three different DSB repair reporter systems, which contained the endonuclease site in the active thymidine kinase gene (TK) locus, we demonstrated separate dose-dependent effects of NSD2 on homologous recombination (HR), canonical-non-homologous end joining (c-NHEJ), and non-canonical-NHEJ (non-c-NHEJ). Endogenous NSD2 has a role in repressing non-c-NHEJ, without affecting DSB repair efficiency by HR or total NHEJ. Furthermore, overexpression of NSD2 promotes c-NHEJ repair and suppresses HR repair. Therefore, we propose that NSD2 has functions in chromatin integrity at the active regions during DSB repair., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Ganciclovir (GCV) is a clinically important drug as it is used to treat viral infections. GCV is incorporated into the DNA during replication, where it interferes with subsequent replication on GCV-incorporated templates. However, the effects of GCV on the host genome and the mechanisms underlying cellular tolerance to GCV remain unclear. In this study, we explored these mechanisms using a collection of mutant DT40 cells. We identified RAD17 /- , BRCA1 -/- , and RAD18 -/- cells as highly GCV-sensitive. RAD17, a component of the alternative checkpoint-clamp loader RAD17-RFC, was required for the activation of the intra-S checkpoint following GCV treatment. BRCA1, a critical factor for promoting homologous recombination (HR), was required for suppressing DNA double-strand breaks (DSBs). Moreover, RAD18, an E3-ligase involved in DNA repair, was critical in suppressing the aberrant ligation of broken chromosomes caused by GCV. We found that BRCA1 suppresses DSBs through HR-mediated repair and template switching (TS)-mediated damage bypass. Moreover, the strong GCV sensitivity of BRCA1 -/- cells was rescued by the loss of 53BP1, despite the only partial restoration in the sister chromatid exchange events which are hallmarks of HR. These results indicate that BRCA1 promotes cellular tolerance to GCV through two mechanisms, TS and HR-mediated repair., (© 2024 The Author(s). Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The fundamental role of cells in safeguarding the genome's integrity against DNA double-strand breaks (DSBs) is crucial for maintaining chromatin homeostasis and the overall genomic stability. Aberrant responses to DNA damage, known as DNA damage responses (DDRs), can result in genomic instability and contribute significantly to tumorigenesis. Unraveling the intricate mechanisms underlying DDRs following severe damage holds the key to identify therapeutic targets for cancer. Chromatin lysine acylation, encompassing diverse modifications such as acetylation, lactylation, crotonylation, succinylation, malonylation, glutarylation, propionylation, and butyrylation, has been extensively studied in the context of DDRs and chromatin homeostasis. Here, we delve into the modifying enzymes and the pivotal roles of lysine acylation and their crosstalk in maintaining chromatin homeostasis and genome integrity in response to DDRs. Moreover, we offer a comprehensive perspective and overview of the latest insights, driven primarily by chromatin acylation modification and associated regulators., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Cells employ diverse strategies to repair double-strand breaks (DSBs), a dangerous form of DNA damage that threatens genome integrity. Eukaryotic nuclei consist of different chromatin environments, each displaying distinct molecular and biophysical properties that can significantly influence the DSB-repair process. DSBs arising in the compact and silenced heterochromatin domains have been found to move to the heterochromatin periphery in mouse and Drosophila to prevent aberrant recombination events. However, it is poorly understood how chromatin components, such as histone post-translational modifications, contribute to these DSB movements within heterochromatin. Using irradiation as well as locus-specific DSB induction in Drosophila tissues and cultured cells, we find enrichment of histone H3 lysine 9 acetylation (H3K9ac) at DSBs in heterochromatin but not euchromatin. We find this increase is mediated by the histone acetyltransferase dGcn5, which rapidly localizes to heterochromatic DSBs. Moreover, we demonstrate that in the absence of dGcn5, heterochromatic DSBs display impaired recruitment of the SUMO E3 ligase Nse2/Qjt and fail to relocate to the heterochromatin periphery to complete repair. In summary, our results reveal a previously unidentified role for dGcn5 and H3K9ac in heterochromatic DSB repair and underscore the importance of differential chromatin responses at heterochromatic and euchromatic DSBs to promote safe repair., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

PARP inhibitors (PARPi) show selective efficacy in tumors with homologous recombination repair (HRR)-defects but the activation mechanism of HRR pathway in PARPi-treated cells remains enigmatic. To unveil it, we searched for the mediator bridging PARP1 to ATM pathways by screening 211 human ubiquitin-related proteins. We discovered TRIM44 as a crucial mediator that recruits the MRN complex to damaged chromatin, independent of PARP1 activity. TRIM44 binds PARP1 and regulates the ubiquitination-PARylation balance of PARP1, which facilitates timely recruitment of the MRN complex for DSB repair. Upon exposure to PARPi, TRIM44 shifts its binding from PARP1 to the MRN complex via its ZnF UBP domain. Knockdown of TRIM44 in cells significantly enhances the sensitivity to olaparib and overcomes the resistance to olaparib induced by 53BP1 deficiency. These observations emphasize the central role of TRIM44 in tethering PARP1 to the ATM-mediated repair pathway. Suppression of TRIM44 may enhance PARPi effectiveness and broaden their use even to HR-proficient tumors., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The master regulator of the DNA damage response, the transcription factor p53, orchestrates multiple downstream responses and coordinates repair processes. In response to double-strand DNA breaks, p53 exhibits pulses of expression, but how it achieves temporal coordination of downstream responses remains unclear. Here, we show that p53's posttranslational modification state is altered between its first and second pulses of expression. We show that acetylations at two sites, K373 and K382, were reduced in the second pulse, and these acetylations differentially affected p53 target genes, resulting in changes in gene expression programs over time. This interplay between dynamics and modification may offer a strategy for cellular hubs like p53 to temporally organize multiple processes in individual cells.

Background: Epigenetic 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., Methods: KDM4B-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., Results: RSK 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., Conclusions: Overall, 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., (© 2024. The Author(s).)

Nucleases are a super family of enzymes that hydrolyze phosphodiester bonds present in genomes. They widely vary in substrates, causing differentiation in cleavage patterns and having a diversified role in maintaining genetic material. Through cellular evolution of prokaryotic to eukaryotic, nucleases become structure-specific in recognizing its own or foreign genomic DNA/RNA configurations as its substrates, including flaps, bubbles, and Holliday junctions. These special structural configurations are commonly found as intermediates in processes like DNA replication, repair, and recombination. The structure-specific nature and diversified functions make them essential to maintaining genome integrity and evolution in normal and cancer cells. In this article, we review their roles in various pathways, including Okazaki fragment maturation during DNA replication, end resection in homology-directed recombination repair of DNA double-strand breaks, DNA excision repair and apoptosis DNA fragmentation in response to exogenous DNA damage, and HIV life cycle. As the nucleases serve as key points for the DNA dynamics, cellular apoptosis, and cancer cell survival pathways, we discuss the efforts in the field in developing the therapeutic regimens, taking advantage of recently available knowledge of their diversified structures and functions., (© The Author(s) (2024). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

This study aimed to investigate whether the plant DNA damage levels and DNA damage response (DDR) are regulated during Agrobacterium infection and potentially manipulated by Agrobacterium to facilitate T-DNA integration. We investigated the plant genomic response to Agrobacterium infection by measuring gamma H2AX levels, which reflect the levels of double-strand DNA breaks (DSBs), and by characterizing transcription of three major DNA repair marker genes NAC82, KU70, and AGO2. These experiments revealed that, globally, Agrobacterium infection did not result in a major increase in DSB content in the host genome. The transcription of the DNA damage repair genes, on the other hand, was elevated upon the wild-type Agrobacterium infection. This transcriptional outcome was largely negated by a mutation in the bacterial virB5 gene which encodes the virulence (Vir) protein B5, a minor component of Agrobacterium pilus necessary for the translocation of Vir effector proteins into the host cell, suggesting that the transcriptional activation of the cellular DNA damage repair machinery requires the transport into the host cell of the Agrobacterium effectors, i.e., the VirD2, VirD5, VirE2, VirE3, and VirF proteins. Most likely, a combination of several of these Vir effectors is required to activate the host DNA repair as their individual loss- or gain-of-function mutants did not significantly affect this process., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Chromothripsis describes the catastrophic shattering of mis-segregated chromosomes trapped within micronuclei. Although micronuclei accumulate DNA double-strand breaks and replication defects throughout interphase, how chromosomes undergo shattering remains unresolved. Using CRISPR-Cas9 screens, we identify a non-canonical role of the Fanconi anemia (FA) pathway as a driver of chromothripsis. Inactivation of the FA pathway suppresses chromosome shattering during mitosis without impacting interphase-associated defects within micronuclei. Mono-ubiquitination of FANCI-FANCD2 by the FA core complex promotes its mitotic engagement with under-replicated micronuclear chromosomes. The structure-selective SLX4-XPF-ERCC1 endonuclease subsequently induces large-scale nucleolytic cleavage of persistent DNA replication intermediates, which stimulates POLD3-dependent mitotic DNA synthesis to prime shattered fragments for reassembly in the ensuing cell cycle. Notably, FA-pathway-induced chromothripsis generates complex genomic rearrangements and extrachromosomal DNA that confer acquired resistance to anti-cancer therapies. Our findings demonstrate how pathological activation of a central DNA repair mechanism paradoxically triggers cancer genome evolution through chromothripsis., Competing Interests: Declaration of interests R.S.L. has received research funding from Day One Biopharmaceuticals, Inc. and clinical trial funding and honorarium from Pfizer, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Long noncoding (lnc)RNAs emerge as regulators of genome stability. The nuclear-enriched abundant transcript 1 (NEAT1) is overexpressed in many tumors and is responsive to genotoxic stress. However, the mechanism that links NEAT1 to DNA damage response (DDR) is unclear. Here, we investigate the expression, modification, localization, and structure of NEAT1 in response to DNA double-strand breaks (DSBs). DNA damage increases the levels and N6-methyladenosine (m 6 A) marks on NEAT1, which promotes alterations in NEAT1 structure, accumulation of hypermethylated NEAT1 at promoter-associated DSBs, and DSB signaling. The depletion of NEAT1 impairs DSB focus formation and elevates DNA damage. The genome-protective role of NEAT1 is mediated by the RNA methyltransferase 3 (METTL3) and involves the release of the chromodomain helicase DNA binding protein 4 (CHD4) from NEAT1 to fine-tune histone acetylation at DSBs. Our data suggest a direct role for NEAT1 in DDR., (© 2024 Mamontova et al.; Published by Cold Spring Harbor Laboratory Press.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yasmin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging., Competing Interests: AU, TH, JN, SS, SS, AU, TB, MM, BM, ZH, JB, PV No competing interests declared, (© 2023, Urbancokova, Hornofova et al.)

Break-induced replication (BIR) is mutagenic, and thus its use requires tight regulation, yet the underlying mechanisms remain elusive. Here we uncover an important role of 53BP1 in suppressing BIR after end resection at double strand breaks (DSBs), distinct from its end protection activity, providing insight into the mechanisms governing BIR regulation and DSB repair pathway selection. We demonstrate that loss of 53BP1 induces BIR-like hyperrecombination, in a manner dependent on Polα-primase-mediated end fill-in DNA synthesis on single-stranded DNA (ssDNA) overhangs at DSBs, leading to PCNA ubiquitination and PIF1 recruitment to activate BIR. On broken replication forks, where BIR is required for repairing single-ended DSBs (seDSBs), SMARCAD1 displaces 53BP1 to facilitate the localization of ubiquitinated PCNA and PIF1 to DSBs for BIR activation. Hyper BIR associated with 53BP1 deficiency manifests template switching and large deletions, underscoring another aspect of 53BP1 in suppressing genome instability. The synthetic lethal interaction between the 53BP1 and BIR pathways provides opportunities for targeted cancer treatment., (© 2024. The Author(s).)

Heavy water, containing the heavy hydrogen isotope, is toxic to cells, although the underlying mechanism remains incompletely understood. In addition, certain enzymatic proton transfer reactions exhibit kinetic isotope effects attributed to hydrogen isotopes and their temperature dependencies, indicative of quantum tunneling phenomena. However, the correlation between the biological effects of heavy water and the kinetic isotope effects mediated by hydrogen isotopes remains elusive. In this study, we elucidated the kinetic isotope effects arising from hydrogen isotopes of water and their temperature dependencies in vitro, focusing on deacetylation, DNA cleavage, and protein cleavage, which are crucial enzymatic reactions mediated by hydrolysis. Intriguingly, the intracellular isotope effects of heavy water, related to the in vitro kinetic isotope effects, significantly impeded multiple DNA double-strand break repair mechanisms crucial for cell survival. Additionally, heavy water exposure enhanced histone acetylation and associated transcriptional activation in cells, consistent with the in vitro kinetic isotope effects observed in histone deacetylation reactions. Moreover, as observed for the in vitro kinetic isotope effects, the cytotoxic effect on cell proliferation induced by heavy water exhibited temperature-dependency. These findings reveal the substantial impact of heavy water-induced isotope effects on cellular functions governed by hydrolytic enzymatic reactions, potentially mediated by quantum-level mechanisms underlying kinetic isotope effects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yasuda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Constantinou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The increased interest in research on DNA damage in neurodegeneration has created a need for the development of tools dedicated to the analysis of DNA damage in neurons. Double-stranded breaks (DSBs) are among the most detrimental types of DNA damage and have become a subject of intensive research. DSBs result in DNA damage foci, which are detectable with the marker γH2AX. Manual counting of DNA damage foci is challenging and biased, and there is a lack of open-source programs optimized specifically in neurons. Thus, we developed a new, fully automated application, SimplySmart_v1, for DNA damage quantification and optimized its performance specifically in primary neurons cultured in vitro., Results: Compared with control neurons, SimplySmart_v1 accurately identifies the induction of DNA damage with etoposide in primary neurons. It also accurately quantifies DNA damage in the desired fraction of cells and processes a batch of images within a few seconds. SimplySmart_v1 was also capable of quantifying DNA damage effectively regardless of the cell type (neuron or NSC-34). The comparative analysis of SimplySmart_v1 with other open-source tools, such as Fiji, CellProfiler and a focinator, revealed that SimplySmart_v1 is the most 'user-friendly' and the quickest tool among others and provides highly accurate results free of variability between measurements. In the context of neurodegenerative research, SimplySmart_v1 revealed an increase in DNA damage in primary neurons expressing abnormal TAR DNA/RNA binding protein (TDP-43)., Conclusions: These findings showed that SimplySmart_v1 is a new and effective tool for research on DNA damage and can successfully replace other available software., (© 2024. Crown.)

DNA rearrangements, including inversions, translocations, and large insertions/deletions (indels), are crucial for crop evolution, domestication, and improvement. The rearrangements are frequently induced by ion beams via the mis-repair of DNA double-strand breaks (DSBs). Unfortunately, how ion beam-induced DSBs are repaired has not been comprehensively analyzed and the mechanisms underlying DNA rearrangements remain unclear. In this study, clonal sectors originating from single mutated cells in carbon ion-irradiated plants were used for whole-genome sequencing analyses after Arabidopsis seeds and seedlings were irradiated. Comparative analyses of the induced mutations (e.g., size and frequency of indels and microhomology at the junctions of the rearrangements) in the irradiated materials suggested that the broken/rejoined DSB ends were more extensively processed in seedlings than in seeds. A mutation to canonical non-homologous end-joining (c-NHEJ), which is a DSB repair pathway with minimal processing of DSB ends, increased the sensitivity to ion beams more in the seeds than in the seedlings, which was consistent with the junction analysis results, indicative of the minor contribution of c-NHEJ to the carbon ion-induced DSB repair in seedlings. Considering the characteristics of the large templated insertions in irradiated seedlings, ion-beam-induced DSBs in seedlings are likely repaired primarily by a polymerase theta-mediated pathway. Polymerase theta-deficient seedlings were more sensitive to ion beams than the c-NHEJ-deficient seedlings, consistent with this hypothesis. This study revealed the key characteristics of ion beam-induced DSBs and the associated repair mechanisms related to the physiological status of the irradiated materials, with implications for elucidating the occurrence and induction of rearrangements., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Purpose: In head and neck squamous cell carcinoma (HNSCC), early complications of the radiotherapy (RT) are observed from the beginning of the treatment to a few months after its end. During external radiotherapy treatment, several patient-dependent parameters can cause a modification of the dose distribution compared to the planned distribution due to variation in patient positioning, anatomy, or intra-fractional movements for example. To verify these parameters during treatment sessions, one of the most commonly used solutions is the cone-beam computed tomography (CBCT). Nowadays, the use of CBCT may constitutes a significant part of the total dose at the end of treatment (up to 10 cGy per session) and more often the volume irradiated by imaging is larger than the one irradiated by the treatment, leading to unintentional irradiation of nearby organs. In this study, we asked whether the imaging low dose added to a following fraction dose (2Gy) may affect the biological response in terms of DNA repair., Material and Methods: Using an IVInomad dosimeter and scintillating fiber probes specially designed for this exploratory study, we exposed fibroblasts cells from head and neck cancer (HNC) patients to a CBCT dose followed by a radiotherapy fraction dose. DNA double strand breaks and DNA repair were assessed by immunofluorescence using the biomarkers gamma H2AX (γH2AX) and pATM., Results: The median dose of CBCT was measured between 17 to 21 mGy per session. The kinetics of both biomarkers were found to be strongly dependent on the individual factor in radiosensitive patients. For HNC patients, a prior CBCT dose applied few minutes before the 2Gy dose may have a sublinear effect on the DNA repair mechanisms and potentially on observed health tissue toxicity., Conclusion: The preliminary results obtained highlight the importance of individual and tissue factors for recognizing and repairing DSB during a treatment by radiotherapy using CBCT., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The licensing step of DNA double-strand break repair by homologous recombination entails resection of DNA ends to generate a single-stranded DNA template for assembly of the repair machinery consisting of the RAD51 recombinase and ancillary factors 1 . DNA end resection is mechanistically intricate and reliant on the tumour suppressor complex BRCA1-BARD1 (ref. 2 ). Specifically, three distinct nuclease entities-the 5'-3' exonuclease EXO1 and heterodimeric complexes of the DNA endonuclease DNA2, with either the BLM or WRN helicase-act in synergy to execute the end resection process 3 . A major question concerns whether BRCA1-BARD1 directly regulates end resection. Here, using highly purified protein factors, we provide evidence that BRCA1-BARD1 physically interacts with EXO1, BLM and WRN. Importantly, with reconstituted biochemical systems and a single-molecule analytical tool, we show that BRCA1-BARD1 upregulates the activity of all three resection pathways. We also demonstrate that BRCA1 and BARD1 harbour stand-alone modules that contribute to the overall functionality of BRCA1-BARD1. Moreover, analysis of a BARD1 mutant impaired in DNA binding shows the importance of this BARD1 attribute in end resection, both in vitro and in cells. Thus, BRCA1-BARD1 enhances the efficiency of all three long-range DNA end resection pathways during homologous recombination in human cells., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5'-terminated strands at DSB sites 1,2 . The breast cancer suppressor BRCA1-BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress 1,3-9 . BRCA1-BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear 10-16 . Using purified recombinant proteins, we show here that BRCA1-BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1-BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11-RAD50-NBS1 and phosphorylated CtIP, BRCA1-BARD1 forms the BRCA1-C complex 17,18 , which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1-C complex 19-21 , inhibits resection, showing that BRCA1-C is a functionally integrated ensemble. Whereas BRCA1-BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4-6,8 ). We show that in the presence of RAD51, BRCA1-BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1-BARD1 in various physiological contexts., (© 2024. The Author(s).)

Break-induced replication (BIR) is a homologous recombination (HR) pathway that repairs one-ended DNA double-strand breaks (DSBs), which can result from replication fork collapse, telomere erosion, and other events. Eukaryotic BIR has been mainly investigated in yeast, where it is initiated by invasion of the broken DNA end into a homologous sequence, followed by extensive replication synthesis proceeding to the chromosome end. Multiple recent studies have described BIR in mammalian cells, the properties of which show many similarities to yeast BIR. While HR is considered as "error-free" mechanism, BIR is highly mutagenic and frequently leads to chromosomal rearrangements-genetic instabilities known to promote human disease. In addition, it is now recognized that BIR is highly stimulated by replication stress (RS), including RS constantly present in cancer cells, implicating BIR as a contributor to cancer genesis and progression. Here, we discuss the past and current findings related to the mechanism of BIR, the association of BIR with replication stress, and the destabilizing effects of BIR on the eukaryotic genome. Finally, we consider the potential for exploiting the BIR machinery to develop anti-cancer therapeutics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

HerA is an ATP-dependent translocase that is widely distributed in archaea and some bacteria. It belongs to the HerA/FtsK translocase bacterial family, which is a subdivision of the RecA family. Currently, it is identified that HerA participates in the repair of DNA double-strand breaks (DSBs) or confers anti-phage defense by assembling other proteins into large complexes. In recent years, there has been a growing understanding of the bioinformatics, biochemistry, structure, and function of HerA subfamily members in both archaea and bacteria. This comprehensive review compares the structural disparities among diverse HerAs and elucidates their respective roles in specific life processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type-II topoisomerase-like protein that becomes covalently linked to DSB ends. Whilst Spo11 oligos-the products of nucleolytic removal by Mre11-have been detected in several organisms, the lifetime of the covalent Spo11-DSB precursor has not been determined and may be subject to alternative processing. Here, we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2-a protein known to repair DNA ends arising from abortive topoisomerase activity-on Spo11 DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 peptides from ssDNA oligos and dsDNA ends even in the presence of competitor genomic DNA. Interestingly, TDP2-processed DSB ends are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate HR at Spo11 DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 expression in meiotic cells was unable to remove Spo11 in vivo-contrasting its ability to aid repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher-order protein complexes at sites of meiotic recombination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The substrate for the repair of DNA damage in living cells is not DNA but chromatin. Chromatin bears a range of modifications, which in turn bind ligands that compact or open chromatin structure, and determine its spatial organization within the nucleus. In some cases, RNA in the form of RNA:DNA hybrids or R-loops modulates DNA accessibility. Each of these parameters can favor particular pathways of repair. Chromatin or nucleosome remodelers are key regulators of chromatin structure, and a number of remodeling complexes are implicated in DNA repair. We cover novel insights into the impact of chromatin structure, nuclear organization, R-loop formation, nuclear actin, and nucleosome remodelers in DNA double-strand break repair, focusing on factors that alter repair functional upon ablation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The DNA dependent protein kinase (DNA-PK) initiates non-homologous recombination (NHEJ), the predominate DNA double-strand break (DSBR) pathway in higher vertebrates. It has been known for decades that the enzymatic activity of DNA-PK [that requires its three component polypeptides, Ku70, Ku80 (that comprise the DNA-end binding Ku heterodimer), and the catalytic subunit (DNA-PKcs)] is present in humans at 10-50 times the level observed in other mammals. Here, we show that the high level of DNA-PKcs protein expression appears evolutionarily in mammals between prosimians and higher primates. Moreover, the RNAs encoding the three component polypeptides of DNA-PK are present at similarly high levels in hominids, new-, and old-world monkeys, but expression of these RNAs in prosimians is ∼5-50 fold less, analogous to the levels observed in other non-primate species. This is reminiscent of the appearance of Alu repeats in primate genomes -- abundant in higher primates, but present at much lower density in prosimians. Alu repeats are well-known for their capacity to promote non-allelic homologous recombination (NAHR) a process known to be inhibited by DNA-PK. Nanopore sequence analyses of cultured cells proficient or deficient in DNA-PK revealed an increase of inter-chromosomal translocations caused by NAHR. Although the high levels of DNA-PK in primates may have many functions, we posit that high levels of DNA-PK may function to restrain deleterious NAHR events between Alu elements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways., Competing Interests: Declaration of competing interest The authors declare no competing interests. Kexin Yang: wrote the manuscript and gathered the document. Lihua Zhu and Chang Liu: modified and edited of the manuscript. Zhu Zhu and Dayang Zhou: collected the literatures. Wenliang Li and Ning Xu: gave direction and reviewed., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background Information: Arpin, an Arp2/3 inhibitory protein, inhibits lamellipodial protrusions and cell migration. Arpin expression is lost in tumor cells of several cancer types., Results: Here we analyzed expression levels of Arpin and various markers using Reverse Phase Protein Array (RPPA) in human mammary carcinomas. We found that Arpin protein levels were correlated with those of several DNA damage response markers. Arpin-null cells display enhanced clustering of double stand breaks (DSBs) when cells are treated with a DNA damaging agent, in line with a previously described role of the Arp2/3 complex in promoting DSB clustering for homologous DNA repair (HDR) in the nucleus. Using a specific HDR assay, we further showed that Arpin depletion increased HDR efficiency two-fold through its ability to inactivate the Arp2/3 complex., Conclusions: Arpin regulates both cell migration in the cytosol and HDR in the nucleus., Significance: Loss of Arpin expression coordinates enhanced cell migration with up-regulated DNA repair, which is required when DNA damage is induced by active cell migration., (© 2024 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Tudor Interacting Repair Regulator (TIRR) is an RNA-binding protein (RBP) that interacts directly with 53BP1, restricting its access to DNA double-strand breaks (DSBs) and its association with p53. We utilized iCLIP to identify RNAs that directly bind to TIRR within cells, identifying the long non-coding RNA NEAT1 as the primary RNA partner. The high affinity of TIRR for NEAT1 is due to prevalent G-rich motifs in the short isoform (NEAT1_1) region of NEAT1. This interaction destabilizes the TIRR/53BP1 complex, promoting 53BP1's function. NEAT1_1 is enriched during the G1 phase of the cell cycle, thereby ensuring that TIRR-dependent inhibition of 53BP1's function is cell cycle-dependent. TDP-43, an RBP that is implicated in neurodegenerative diseases, modulates the TIRR/53BP1 complex by promoting the production of the NEAT1 short isoform, NEAT1_1. Together, we infer that NEAT1_1, and factors regulating NEAT1_1, may impact 53BP1-dependent DNA repair processes, with implications for a spectrum of diseases., (© 2024. The Author(s).)

Targeting DNA repair pathways is an important strategy in anticancer therapy. However, the unrevealed interactions between different DNA repair systems may interfere with the desired therapeutic effect. Among DNA repair systems, BER and NHEJ protect genome integrity through the entire cell cycle. BER is involved in the repair of DNA base lesions and DNA single-strand breaks (SSBs), while NHEJ is responsible for the repair of DNA double-strand breaks (DSBs). Previously, we showed that BER deficiency leads to downregulation of NHEJ gene expression. Here, we studied BER's response to NHEJ deficiency induced by knockdown of NHEJ scaffold protein XRCC4 and compared the knockdown effects in normal (TIG-1) and hTERT-modified cells (NBE1). We investigated the expression of the XRCC1 , LIG3, and APE1 genes of BER and LIG4 ; the Ku70 / Ku80 genes of NHEJ at the mRNA and protein levels; as well as p53 , Sp1 and PARP1 . We found that, in both cell lines, XRCC4 knockdown leads to a decrease in the mRNA levels of both BER and NHEJ genes, though the effect on protein level is not uniform. XRCC4 knockdown caused an increase in p53 and Sp1 proteins, but caused G1/S delay only in normal cells. Despite the increased p53 protein, p21 did not significantly increase in NBE1 cells with overexpressed hTERT, and this correlated with the absence of G1/S delay in these cells. The data highlight the regulatory function of the XRCC4 scaffold protein and imply its connection to a transcriptional regulatory network or mRNA metabolism.

Topoisomerases alter DNA topology by making transient DNA strand breaks (DSBs) in DNA. The DNA cleavage reaction mechanism includes the formation of a reversible protein/DNA complex that allows rapid resealing of the transient break. This mechanism allows changes in DNA topology with minimal risks of persistent DNA damage. Nonetheless, small molecules, alternate DNA structures, or mutations in topoisomerase proteins can impede the resealing of the transient breaks, leading to genome instability and potentially cell death. The consequences of high levels of enzyme/DNA adducts differ for type I and type II topoisomerases. Top1 action on DNA containing ribonucleotides leads to 2-5 nucleotide deletions in repeated sequences, while mutant Top1 enzymes can generate large deletions. By contrast, small molecules that target Top2, or mutant Top2 enzymes with elevated levels of cleavage lead to small de novo duplications. Both Top1 and Top2 have the potential to generate large rearrangements and translocations. Thus, genome instability due to topoisomerase mis-function is a potential pathogenic mechanism especially leading to oncogenic progression. Recent studies support the potential roles of topoisomerases in genetic changes in cancer cells, highlighting the need to understand how cells limit genome instability induced by topoisomerases. This review highlights recent studies that bear on these questions.