Luminal progenitor and mature cells are more susceptible than basal cells to radiation-induced DNA double-strand breaks in rat mammary tissue.

Ionizing radiation promotes mammary carcinogenesis. Induction of DNA double-strand breaks (DSBs) is the initial event after radiation exposure, which can potentially lead to carcinogenesis, but the dynamics of DSB induction and repair are not well understood at the tissue level. In this study, we used female rats, which have been recognized as a useful experimental model for studying radiation effects on the mammary gland. We focused on differences in DSB kinetics among basal cells, luminal progenitor and mature cells in different parts of the mammary duct. 53BP1 foci were used as surrogate markers of DSBs, and 53BP1 foci in each mammary epithelial cell in immunostained tissue sections were counted 1-24 h after irradiation and fitted to an exponential function of time. Basal cells were identified as cytokeratin (CK) 14+ cells, luminal progenitor cells as CK8 + 18low cells and luminal mature cells as CK8 + 18high cells. The number of DSBs per nucleus tended to be higher in luminal cells than basal cells at 1 h post-irradiation. A model analysis indicated that basal cells in terminal end buds (TEBs), which constitute the leading edge of the mammary duct, had significantly fewer initial DSBs than the two types of luminal cells, and there was no significant difference in initial amount among the cell types in the subtending duct. The repair rate did not differ among mammary epithelial cell types or their locations. Thus, luminal progenitor and mature cells are more susceptible to radiation-induced DSBs than are basal cells in TEBs.

Dose-dependent effects of histone methyltransferase NSD2 on site-specific double-strand break repair.

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.

The MRE11-RAD50-NBS1 complex both starts and extends DNA end resection in mouse meiosis.

Nucleolytic resection of DNA ends is critical for homologous recombination, but its mechanism is not fully understood, particularly in mammalian meiosis. Here we examine roles of the conserved MRN complex (MRE11, RAD50, and NBS1) through genome-wide analysis of meiotic resection in mice with various MRN mutations, including several that cause chromosomal instability in humans. Meiotic DSBs form at elevated levels but remain unresected if Mre11 is conditionally deleted, thus MRN is required for both resection initiation and regulation of DSB numbers. Resection lengths are reduced to varying degrees in MRN hypomorphs or if MRE11 nuclease activity is attenuated in a conditional nuclease-dead Mre11 model. These findings unexpectedly establish that MRN is needed for longer-range extension of resection, not just resection initiation. Finally, resection defects are additively worsened by combining MRN and Exo1 mutations, and mice that are unable to initiate resection or have greatly curtailed resection lengths experience catastrophic spermatogenic failure. Our results elucidate multiple functions of MRN in meiotic recombination, uncover unanticipated relationships between short- and long-range resection, and establish the importance of resection for mammalian meiosis.

Synergistic Effects of Melatonin on Radiosensitization in Carbon-ion Radiotherapy.

BACKGROUND/AIM: Despite the established antitumor effectiveness and synergistic interactions of melatonin with photon irradiation, its role in carbon-ion radiotherapy remains uncertain. This study aimed to elucidate the mechanisms and potential clinical advantages of combining exogenous melatonin therapy with carbon-ion radiotherapy. MATERIALS AND METHODS: The investigation assessed the impact of combining exogenous melatonin with photon or carbon-ion irradiation on cell-cycle modulation and DNA-repair capability using the melanoma cell line B16F10. RNA sequencing and bioinformatics analysis were conducted to explore mechanisms and evaluate potential clinical benefits, with validation performed on the osteosarcoma cell line LM8. RESULTS: Pre-treatment with melatonin reduced the survival fraction of B16F10 and LM8 cells upon exposure to photon and carbon-ion radiation. Mechanistically, melatonin was found to inhibit G2/M arrest, preserve DNA damage, and suppress key genes involved in DNA double-strand break repair after 8 Gy carbon-ion radiation. Furthermore, RNA sequencing and bioinformatics analysis revealed favorable changes in genes associated with survival and metastasis, highlighting potential clinical significance. LM8 cells treated with melatonin exhibited increased radiosensitivity and suppression of DNA-repair proteins. CONCLUSION: The combination of exogenous melatonin not only heightened radiosensitivity and modulated hallmark tumor gene sets in vitro but also markedly suppressed the efficiency of DNA double-strand break-repair pathway, thus enhancing the cytotoxicity of carbon-ion radiotherapy.

Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair.

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

Current status and prospect of the DNA double-strand break repair pathway in colorectal cancer development and treatment.

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.

The DNA double-strand break repair proteins γH2AX, RAD51, BRCA1, RPA70, KU80, and XRCC4 exhibit follicle-specific expression differences in the postnatal mouse ovaries from early to older ages.

PURPOSE: Ovarian aging is closely related to a decrease in follicular reserve and oocyte quality. The precise molecular mechanisms underlying these reductions have yet to be fully elucidated. Herein, we examine spatiotemporal distribution of key proteins responsible for DNA double-strand break (DSB) repair in ovaries from early to older ages. Functional studies have shown that the γH2AX, RAD51, BRCA1, and RPA70 proteins play indispensable roles in HR-based repair pathway, while the KU80 and XRCC4 proteins are essential for successfully operating cNHEJ pathway. METHODS: Female Balb/C mice were divided into five groups as follows: Prepuberty (3 weeks old; n = 6), puberty (7 weeks old; n = 7), postpuberty (18 weeks old; n = 7), early aged (52 weeks old; n = 7), and late aged (60 weeks old; n = 7). The expression of DSB repair proteins, cellular senescence (ß-GAL) and apoptosis (cCASP3) markers was evaluated in the ovaries using immunohistochemistry. RESULT: ß-GAL and cCASP3 levels progressively increased from prepuberty to aged groups (P < 0.05). Notably, γH2AX levels varied in preantral and antral follicles among the groups (P < 0.05). In aged groups, RAD51, BRCA1, KU80, and XRCC4 levels increased (P < 0.05), while RPA70 levels decreased (P < 0.05) compared to the other groups. CONCLUSIONS: The observed alterations were primarily attributed to altered expression in oocytes and granulosa cells of the follicles and other ovarian cells. As a result, the findings indicate that these DSB repair proteins may play a role in the repair processes and even other related cellular events in ovarian cells from early to older ages.

Progerin Can Induce DNA Damage in the Absence of Global Changes in Replication or Cell Proliferation.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.

Arabidopsis cryptochromes interact with SOG1 to promote the repair of DNA double-strand breaks.

Cryptochromes (CRYs) are blue light (BL) photoreceptors to regulate a variety of physiological processes including DNA double-strand break (DSB) repair. SUPPRESSOR OF GAMMA RADIATION 1 (SOG1) acts as the central transcription factor of DNA damage response (DDR) to induce the transcription of downstream genes, including DSB repair-related genes BRCA1 and RAD51. Whether CRYs regulate DSB repair by directly modulating SOG1 is unknown. Here, we demonstrate that CRYs physically interact with SOG1. Disruption of CRYs and SOG1 leads to increased sensitivity to DSBs and reduced DSB repair-related genes' expression under BL. Moreover, we found that CRY1 enhances SOG1's transcription activation of DSB repair-related gene BRCA1. These results suggest that the mechanism by which CRYs promote DSB repair involves positive regulation of SOG1's transcription of its target genes, which is likely mediated by CRYs-SOG1 interaction.

KDM4B mutations in human cancers.

Homologous recombination (HR) is essential for repair of DNA double-strand breaks (DSBs) and restart of stalled or collapsed replication forks. Most cancers are characterized by mutations in components of the DSB repair pathways. Redundant DSB repair pathways exist in eukaryotes from yeast to humans and recent evidence has shown that complete loss of HR function appears to be lethal. Recent evidence has also shown that cancer cells with mutations in one DSB repair pathway can be killed by inhibiting one or more parallel pathways, a strategy that is currently aggressively explored as a cancer therapy. KDM4B is a histone demethylase with pleiotropic functions, which participates in preparing DSBs for repair by contributing to chromatin remodeling. In this report we carried out a pan-cancer analysis of KDM4B mutations with the goal of understanding their distribution and interaction with other DSB genes. We find that although KDM4B mutations co-occur with DSB repair genes, most KDM4B mutations are not drivers or pathogenic. A sequence conservation analysis from yeast to humans shows that highly conserved residues are resistant to mutation. Finally, all mutations occur in a heterozygous state. A single mutation, R986L, was predicted to significantly affect protein structure using computational modeling. This analysis suggests that KDM4B makes contributions to DSB repair but is not a key player.

Functional genetic variants of GEN1 predict overall survival of Chinese epithelial ovarian cancer patients.

BACKGROUND: Inherited variations in DNA double-strand break (DSB) repair pathway are known to influence ovarian cancer occurrence, progression and treatment response. Despite its significance, survival-associated genetic variants within the DSB pathway remain underexplored. METHODS: In the present study, we performed a two-phase analysis of 19,290 single-nucleotide polymorphisms (SNPs) in 199 genes in the DSB repair pathway from a genome-wide association study (GWAS) dataset and explored their associations with overall survival (OS) in 1039 Han Chinese epithelial ovarian carcinoma (EOC) patients. After utilizing multivariate Cox regression analysis with bayesian false-discovery probability for multiple test correction, significant genetic variations were identified and subsequently underwent functional prediction and validation. RESULTS: We discovered a significant association between poor overall survival and the functional variant GEN1 rs56070363 C > T (CT + TT vs. TT, adjusted hazard ratio (HR) = 2.50, P < 0.001). And the impact of GEN1 rs56070363 C > T on survival was attributed to its reduced binding affinity to hsa-miR-1287-5p and the resultant upregulation of GEN1 mRNA expression. Overexpression of GEN1 aggregated EOC cell proliferation, invasion and migration presumably by influencing the expression of immune inhibitory factors, thereby elevating the proportion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and then constructing an immunosuppressive tumor microenvironment. CONCLUSIONS: In conclusion, GEN1 rs56070363 variant could serve as a potential predictive biomarker and chemotherapeutic target for improving the survival of EOC patients.

Acute particulate hexavalent chromium exposure induces DNA double-strand breaks and activates homologous recombination repair in rat lung tissue.

Hexavalent chromium [Cr(VI)] is an established human lung carcinogen, but the carcinogenesis mechanism is poorly understood. Chromosome instability, a hallmark of lung cancer, is considered a major driver of Cr(VI)-induced lung cancer. Unrepaired DNA double-strand breaks are the underlying cause, and homologous recombination repair is the primary mechanism preventing Cr(VI)-induced DNA breaks from causing chromosome instability. Cell culture studies show acute Cr(VI) exposure causes DNA double-strand breaks and increases homologous recombination repair activity. However, the ability of Cr(VI)-induced DNA breaks and repair impact has only been reported in cell culture studies. Therefore, we investigated whether acute Cr(VI) exposure could induce breaks and homologous recombination repair in rat lungs. Male and female Wistar rats were acutely exposed to either zinc chromate particles in a saline solution or saline alone by oropharyngeal aspiration. This exposure route resulted in increased Cr levels in each lobe of the lung. We found Cr(VI) induced DNA double-strand breaks in a concentration-dependent manner, with females being more susceptible than males, and induced homologous recombination repair at similar levels in both sexes. Thus, these data show this driving mechanism discovered in cell culture indeed translates to lung tissue in vivo.

PARP inhibitor synthetic lethality in ATM biallelic mutant cancer cell lines is associated with BRCA1/2 and RAD51 downregulation.

Background: Ataxia telangiectasia-mutated (ATM) kinase is a central regulator of the DNA damage response (DDR) signaling pathway, and its function is critical for the maintenance of genomic stability in cells that coordinate a network of cellular processes, including DNA replication, DNA repair, and cell cycle progression. ATM is frequently mutated in human cancers, and approximately 3% of lung cancers have biallelic mutations in ATM, i.e., including 3.5% of lung adenocarcinomas (LUAD) and 1.4% of lung squamous cell carcinomas (LUSC). Methods: We investigated the potential of targeting the DDR pathway in lung cancer as a potential therapeutic approach. In this context, we examined whether ATM loss is synthetically lethal with niraparib monotherapy. This exploration involved the use of hATM knockout (KO) isogenic cell lines containing hATM homozygous (-/-) and heterozygous (+/-) generated via CRISPR/Cas9 gene knockout technology in DLD-1, a human colorectal adenocarcinoma cell line. Subsequently, we extended our investigation to non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) models for further validation of poly ADP-ribose polymerase inhibitor (PARPi) synthetic lethality in ATM mutant NSCLC models. Results: Here, we demonstared that biallelic hATM deletion (-/-) in DLD-1 impairs homologous recombination (HR) repair function and sensitizes cells to the PARPi, niraparib. Niraparib also caused significant tumor regression in one-third of the NSCLC PDX models harboring deleterious biallelic ATM mutations. Loss of hATM (-/-) was concomitantly associated with low BRCA1 and BRCA2 protein expression in both the hATM (-/-) DLD-1 cell line and PARPi-sensitive ATM mutant NSCLC PDX models, suggesting a downstream effect on the impairment of HR-mediated DNA checkpoint signaling. Further analysis revealed that loss of ATM led to inhibition of phosphorylation of MRN (Mre11-Rad50-NBS1) complex proteins, which are required for ATM-mediated downstream phosphorylation of p53, BRCA1, and CHK2. Conclusions: Taken together, our findings highlight that the synthetic lethality of niraparib in ATM-deficient tumors can be regulated through a subsequent effect on the modulation of BRCA1/2 expression and its effect on HR function.

DNA double-strand break repair capacity and its pathway gene variants predict the risk and prognosis of lung cancer.

OBJECTIVES: This study aims to investigate the association between DNA double-strand breaks (DSBs) repair capacity, variations in DSBs-related genes, and the occurrence and prognosis of lung cancer in the Chinese population. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from 98 lung cancer patients and 60 healthy individuals. The individual DSBs repair capacity was assessed by measuring changes in γ-H2AX levels after treatment with etoposide. Exonic sequencing of 45 DSBs-related genes was performed on PBMC DNA. Logistic regression analysis was conducted to examine the relationship between lung cancer risk and DSBs repair capacity as well as germlines gene variations. Survival analysis employed the Cox proportional hazards regression model, Kaplan-Meier method, and Log-rank test. RESULTS: Lower DSBs repair capacity predicted an increased risk of developing lung cancer (OR = 0.94, 95 %CI = 0.917-0.964, P＜0.001). Among lung cancer patients, higher DSBs repair capacity was associated with shorter progression-free survival (PFS) during first-line treatment (HR = 1.80, 95 %CI = 1.10-3.00, P = 0.031). Patients with BRCA1 mutations had shorter overall survival (OS) (HR = 1.92, 95 %CI = 1.12-3.28, P = 0.018). Patients with FOXO3 mutations had shorter PFS (HR = 4.23, 95 %CI = 1.44-12.36, P = 0.009). Analysis of patients treated with immune checkpoint inhibitors (ICIs) indicated that LIG4 mutations were associated with shorter PFS (HR = 2.90, 95 %CI = 1.00-8.10, P = 0.041). CONCLUSIONS: This study concludes that assessing DSBs repair capacity holds promise for predicting both lung cancer risk and prognosis in the Chinese population. Further large-scale studies and functional validation of specific gene mutations related to double-strand breaks are necessary for confirmation.

(Single-stranded DNA) gaps in understanding BRCAness.

The tumour-suppressive roles of BRCA1 and 2 have been attributed to three seemingly distinct functions - homologous recombination, replication fork protection, and single-stranded (ss)DNA gap suppression - and their relative importance is under debate. In this review, we examine the origin and resolution of ssDNA gaps and discuss the recent advances in understanding the role of BRCA1/2 in gap suppression. There are ample data showing that gap accumulation in BRCA1/2-deficient cells is linked to genomic instability and chemosensitivity. However, it remains unclear whether there is a causative role and the function of BRCA1/2 in gap suppression cannot unambiguously be dissected from their other functions. We therefore conclude that the three functions of BRCA1 and 2 are closely intertwined and not mutually exclusive.

Exploring factors influencing choice of DNA double-strand break repair pathways.

DNA double-strand breaks (DSBs) represent one of the most severe threats to genomic integrity, demanding intricate repair mechanisms within eukaryotic cells. A diverse array of factors orchestrates the complex choreography of DSB signaling and repair, encompassing repair pathways, such as non-homologous end-joining, homologous recombination, and polymerase-θ-mediated end-joining. This review looks into the intricate decision-making processes guiding eukaryotic cells towards a particular repair pathway, particularly emphasizing the processing of two-ended DSBs. Furthermore, we elucidate the transformative role of Cas9, a site-specific endonuclease, in revolutionizing our comprehension of DNA DSB repair dynamics. Additionally, we explore the burgeoning potential of Cas9's remarkable ability to induce sequence-specific DSBs, offering a promising avenue for precise targeting of tumor cells. Through this comprehensive exploration, we unravel the intricate molecular mechanisms of cellular responses to DSBs, shedding light on both fundamental repair processes and cutting-edge therapeutic strategies.

[Research Progress in the Roles of MRE11-RAD50-NBS1 Complex and Human Diseases].

DNA is susceptible to various factors in vitro and in vivo and experience different forms of damage,among which double-strand break(DSB)is a deleterious form.To maintain the stability of genetic information,organisms have developed multiple mechanisms to repair DNA damage.Among these mechanisms,homologous recombination(HR)is praised for the high accuracy.The MRE11-RAD50-NBS1(MRN)complex plays an important role in HR and is conserved across different species.The knowledge on the MRN complex mainly came from the previous studies in Saccharomyces cerevisiae and Caenorhabditis elegans,while studies in the last decades have revealed the role of mammalian MRN complex in DNA repair of higher animals.In this review,we first introduces the MRN complex regarding the composition,structure,and roles in HR.In addition,we discuss the human diseases such as ataxia-telangiectasia-like disorder,Nijmegen breakage syndrome,and Nijmegen breakage syndrome-like disorder that are caused by dysfunctions in the MRN complex.Furthermore,we summarize the mouse models established to study the clinical phenotypes of the above diseases.

Duocarmycin SA Reduces Proliferation and Increases Apoptosis in Acute Myeloid Leukemia Cells In Vitro.

Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by an expansion of immature myeloid precursors. Despite therapeutic advances, the prognosis of AML patients remains poor and there is a need for the evaluation of promising therapeutic candidates to treat the disease. The objective of this study was to evaluate the efficacy of duocarmycin Stable A (DSA) in AML cells in vitro. We hypothesized that DSA would induce DNA damage in the form of DNA double-strand breaks (DSBs) and exert cytotoxic effects on AML cells within the picomolar range. Human AML cell lines Molm-14 and HL-60 were used to perform 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), DNA DSBs, cell cycle, 5-ethynyl-2-deoxyuridine (EdU), colony formation unit (CFU), Annexin V, RNA sequencing and other assays described in this study. Our results showed that DSA induced DNA DSBs, induced cell cycle arrest at the G2M phase, reduced proliferation and increased apoptosis in AML cells. Additionally, RNA sequencing results showed that DSA regulates genes that are associated with cellular processes such as DNA repair, G2M checkpoint and apoptosis. These results suggest that DSA is efficacious in AML cells and is therefore a promising potential therapeutic candidate that can be further evaluated for the treatment of AML.

End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well-characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the nematode Ascaris to study the timing of PDE breaks and examine the DSBs and their end processing. Using END-seq, we characterize the DSB ends and demonstrate that DNA breaks are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3' overhangs before the addition of telomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends in Ascaris may be due to the sequestration of the eliminated DNA into micronuclei, preventing their ends from telomere healing. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for nematode PDE.