In meiosis, homologous recombination (HR) facilitates the halving of genomic content in diploid parents to produce haploid gametes, while also reshuffling genetic material. However, studies in yeast and mammals have suggested that HR during meiosis also makes the process mutagenic, and it has been proposed that the single-strand (ss) DNA formed by HR of programmed double-strand breaks (DSBs) contributes to the observed mutagenesis. To determine the full mutagenic potential of ssDNA formed during meiosis, we expressed human APOBEC3A (A3A), a deaminase that specifically attacks and therefore allows detection of ssDNA, in meiotic yeast cells. We demonstrate that meiotic cells accumulate long tracts of ssDNA manifested by A3A-mutation clusters, of up to 134 mutations spanning more than 25 kb. We show that the formation of mutation clusters during meiosis require Spo11-induced DSBs, and that break-induced replication and hyper-resection of DSBs are the primary mechanisms underlying the formation of long ssDNA. We also report meiotic ssDNA accumulation in promoters and tRNA genes, revealing them as additional sources of mutagenesis during meiosis. Together, our results demonstrate the high mutagenic potential of meiosis and provide insight into mechanisms that can fuel evolution and promote congenital diseases in humans.

Toxicogenomic assessment of 137Cs in zebrafish embryos across developmental stages using an AOP network approach.

PCNA loading and stabilization at primer-template junctions is crucial for processive DNA synthesis by replicative polymerases. It is also essential for strand displacement synthesis during Break Induced Replication (BIR). In this study, we employed a single molecule approach to directly visualize the role of PCNA, DNA polymerase δ and the Pif1 DNA helicase in these pathways. We first dissected the preferential loading of PCNA by RFC to 3’-junctions of gaps, nicks and flapped substrates. Moreover, we found that Pol δ and Pif1 both stabilize loaded PCNA and reduce its diffusion coefficient along DNA. We further show that the interaction of PCNA with Pol δ is essential for gap-filling synthesis. On a 5’-flapped substrate, we show that both Pol δ and Pif1 are required for strand displacement synthesis, while PCNA is not; although, it stimulates the process.

Gross chromosomal rearrangements are a hallmark of many diseases and cancers. The study of their biogenesis and the mechanisms underlying their formation is greatly facilitated by the availability of genetic reporter assays in model organisms. We present here a novel GCR assay developed in fission yeast, a highly relevant model for understanding genome instability related to human biology. The reporter employs canavanine counter-selection to detect GCRs within a chromosomal context. Using this assay, we identified natural hotspots for GCRs, including inverted long terminal repeats (IR-LTRs). Structural analysis of GCR events showed that IR-LTR-induced GCRs mainly result in either terminal deletions with adjacent inverted duplications or repair via long-range break-induced replication (BIR). Deleting IR-LTRs reduces the GCR rate and reveals another hotspot driven by BIR between homeologous aldo/keto reductase genes on opposite arms of chromosome I. This is the first evidence that BIR can occur in S. pombe on long tracks reaching up to 600 kb. Besides highlighting genome rearrangement hotspots, the assay also identifies regulators of genome instability in fission yeast. Loss of Nup132, a component of the nuclear pore complex, increases IR-LTRs-induced GCRs, while the budding yeast homolog Nup133 has no effect on the stability of a structurally similar IR. In contrast, disrupting djc9 , which encodes a conserved histone H3-H4 binding protein, decreases GCR rates. Overall, this sensitive GCR assay enables the identification of factors that control spontaneous and fragile motif-induced chromosomal instability, including those conserved in humans but lost through evolution in other organisms.

Alternative lengthening of telomeres (ALT) is a recombination-based pathway enabling cancer cells to maintain telomeres. ALT establishment remains poorly understood due to difficulties identifying its molecular steps. Here, using Oxford Nanopore sequencing and computational modeling, we track the evolution of individual chromosome end structures during ALT establishment in yeast and delineate three molecular milestones. First, homologous recombination via break-induced replication (BIR) at telomeres and sub-telomeric regions delays senescence. Second, BIR interruption and microhomology-mediated recombination promote initial telomere extension and telomeric circle formation. Third, the final extension—critical for chromosome end stabilization—utilizes a highly mutagenic replication mechanism to copy telomeric circles. Linking these newly defined ALT milestones is Mph1, the homolog of human FANCM, which plays important roles throughout ALT establishment by disrupting BIR synthesis and promoting template switching. Our findings support a model where template switching during DNA repair synthesis drives the transitioning through the multiple steps involved in ALT establishment and progression, ultimately producing ALT survivors.

Break-induced replication (BIR) facilitates single-ended DNA double-strand break (DSB) repair. Upon homologous recombination-mediated strand invasion into a homologous repair template, BIR is catalysed by a minimal replisome comprising PCNA, DNA polymerase δ (Pol δ), and the Pif1 helicase. Here, we identify an interaction between Pol δ and single-stranded DNA (ssDNA)-binding protein RPA mediated by an RPA-binding module (RBM) within Pol δ subunit Pol32 and RPA subunit Rfa1. Pol32 RBM phosphorylation at Thr256 and Thr257 increases its affinity for Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in vivo. This suggests that Pol32 functions as a rheostat whose phosphorylation enhances Pol δ’s affinity for RPA-bound BIR intermediates, thereby boosting BIR efficiency. Modelling indicates that Pol32 phospho-RBM-Rfa1 interactions mirror the binding mode of RBMs in Pif1 and the FANCM helicase and BIR antagonist Mph1. This implies a key role for RPA in the dynamic orchestration of the enzymes mediating BIR.

Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replication-associated single-ended DSBs (seDSBs). Here, we demonstrate that microhomology-mediated end joining (MMEJ) acts directly to repair seDSBs at broken replication forks (fork-MMEJ), preferentially on the leading strands, and functions cooperatively with BIR. While promoted by DNA polymerase theta (Polθ), fork-MMEJ operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, distinct from canonical MMEJ (cMMEJ), which is defined at replication-independent double-ended DSBs (deDSBs). ATR, activated as end resection proceeds, serves as a pivotal switch to suppress fork-MMEJ while promoting BIR. The combined inactivation of ATR and Polθ synergistically kills cancer cells under high replication stress with minimal toxicity to normal cells. Together, our study provides fundamental insights into the MMEJ mechanism and offers new strategies for cancer treatment.

Small supernumerary marker chromosomes (sSMCs) remain a diagnostic challenge despite sequencing advances. As the field shifts toward cytogenomics, there is a need to establish methodologies to resolve these complex genetic variants at base pair resolution, as well as to identify their chromosomal origin and formation mechanism. Here, we apply long-read genome sequencing (lrGS) in combination with the telomere-to-telomere (T2T-CHM13) assembly to characterize the structure and genomic content of 10 clinically detected sSMCs. We use sequencing data to reconstruct the derivative chromosomes, identify breakpoint junctions (BPJs), and infer formation mechanisms. We resolve the BPJs of nine of the 10 sSMCs at base pair resolution. The analysis reveals six simple intrachromosomal rearrangements (one continuous and five discontinuous) with one to three BPJs, one complex three-way translocation with two BPJs, and two highly complex intrachromosomal rearrangements with five and nine BPJs, respectively. Breakpoint analysis reveals distinct mechanistic signatures: Simple sSMCs show features consistent with microhomology-mediated end joining (MMEJ) or microhomology-mediated break-induced replication (MMBIR), whereas complex sSMCs demonstrate evidence of translocation, chromoanasynthesis, and breakage–fusion–bridge (BFB) cycles. Haplotype analysis supports trisomy rescue in four cases, including all three complex sSMCs. In summary, our study demonstrates that lrGS combined with T2T-CHM13 enables detailed structural and mechanistic characterization of sSMCs, providing experimental support for disruption of trisomy rescue as a key formation mechanism. This work illustrates the feasibility of resolving highly challenging chromosomal abnormalities using long-read sequencing technologies.

Tandem duplications (TDs) are a common form of genomic rearrangements with both adaptive and pathogenic consequences. While prevalent in genomically unstable cancer genomes, TDs are rarely detected in normal tissues, suggesting the existence of robust protective mechanisms. Here, we identify the histone chaperone TONSL/TONSOKU (tnsl-1 in C. elegans) as a critical suppressor of TD formation. Loss of tnsl-1 results in the accumulation of TDs in two distinct size classes (~25 kb and ~300 kb), arising from different developmental contexts: small TDs emerge in rapidly dividing embryonic cells, whereas large TDs form in slower-dividing germline progenitors. Both classes depend on polymerase theta-mediated end joining (TMEJ), implicating DNA double-strand breaks in their genesis. Inhibition of break-induced replication (BIR) via Pif1 helicase loss reduces TD size, revealing a role for BIR in TD expansion. Remarkably, TONSL-deficient Arabidopsis thaliana exhibit an identical TD signature, highlighting the evolutionary conservation of this genome surveillance mechanism. These findings position TONSL as a cross-kingdom guardian of genome integrity through suppression of TD formation.

Introduction: Most intrachromosomal rearrangements characterized by terminal monosomy and trisomy concomitantly seen in different arms of the same chromosome usually arise due to pericentric inversions in parents. Forty-seven patients have been described with such alteration in chromosome 18. Methods: We investigated a patient with dup(18p)/del(18q) whose rearrangement was characterized by karyotype, catalog and custom chromosomal microarrays, fluorescence in situ hybridization (FISH), whole genome sequencing, and Sanger sequencing. Results: The patient presents a 9.7 Mb terminal duplication in 18p and a 25.8 Mb terminal deletion in 18q. The duplicated segment is inserted into the long arm of chromosome 18 in an inverted position. At the junction point, we found a 19-nucleotide segment insertion derived from a downstream 18q sequence and the presence of microhomology with both the breakpoint from the long-arm region and the one from the inverted duplicated segment from the short arm. Conclusion: We described, to the best of our knowledge, the fifth confirmed case of concomitant terminal trisomy and monosomy in different arms of chromosome 18 which is not due to a pericentric parental inversion, and the first with breakpoints determined at the nucleotide level. The combination of various techniques enabled us to infer the rearrangement’s likely mechanism of formation. The structural features observed are consistent with replication-based mechanisms, including Fork Stalling and Template Switching and Microhomology-Mediated Break-Induced Replication. The detailed rearrangement characterization also allowed for a more detailed karyotype-phenotype correlation, which indicates that the 18q deletion is likely the cause of most of the patient’s phenotypical alterations.

Chromoanasynthesis is a form of complex chromosomal rearrangement (CCR) commonly detected in cancers and congenital disorders, but the mechanism underlying its generation remain elusive. Here we develop a single-molecule long-read DNA sequencing approach to characterise ultra-complex mutational events, consistent with chromoanasynthesis, occurring at shortened telomeres and sub-telomeric DNA double-strand breaks in human cells. Our data reveal that chromoanasynthesis is generated by microhomology-mediated break-induced replication (MM-BIR), occurring specifically in mitosis. Surprisingly, this mitotic pathway involves a collaboration between microhomology-mediated end-joining (MMEJ) and BIR, where MMEJ proteins initiate a Polδ-dependent BIR pathway that is regulated by PIF1, POLD3 and PCNA. This pathway is highly prone to template switching and can generate dramatic amplification of genomic loci in a single event. Our findings help explain the extreme mutagenic nature of chromoanasynthesis and establish mitotic MM-BIR as a key driver of CCRs, with important implications for the origin of cancers and congenital disorders.

Telomeres are protected by the shelterin complex and consist of TTAGGG repeats. Their gradual erosion triggers replicative senescence unless counteracted by telomerase or the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway, utilized by 10-15% of cancers. Accumulating evidence indicates that the decision to activate ALT is intimately linked to the plasticity of telomeric chromatin. This review integrates recent data showing that both constitutive and facultative heterochromatin marks shape ALT activity. We summarize the dynamic regulation of telomeric chromatin and explore the contrasting evidence for two models: the 'open telomeric chromatin model,' where loss of constitutive heterochromatin (e.g., reduced H3K9me3) promotes ALT by increasing chromatin accessibility for homologous recombination (HR) factors, and the 'closed telomeric chromatin model,' where a specific gain of heterochromatic features (e.g., H3K9me3 or H3K27me3) facilitates ALT by creating a specialized phase-separated environment that promotes telomere clustering and break-induced replication (BIR). Resolving this paradox is crucial for understanding ALT initiation and for developing promising synthetic-lethal strategies against ALT-dependent cancers.

Approximately 10-15% of cancers rely on Alternate Lengthening of Telomeres (ALT), which is a telomerase-independent, BIR (Break-Induced Replication)-based pathway for maintaining long-term replicative potential. As ALT is over-represented in mesenchymal-type tumors, we investigated, via RNA-seq, the extent to which the EMT-promoting factor ZEB1 regulates this pathway. The ALT-associated genes targeted by ZEB1, including PML, RMI2, POLD4, RPA3 (induced), SLX4, and WRN (repressed), in the aggregate, suggest that it regulates ALT at multiple steps in that pathway. ZEB1-deficient cells showed a significant reduction in telomere length as well as in two hallmarks of ALT, C-circle levels and the size and number of ALT-associated PML Bodies (APBs), which are the telomere-aggregating compartments in which BIR occurs. As one of the most highly regulated genetic targets of ZEB1 was the pro-epithelial alternative splicing factor ESRP1, we investigated whether the repression of this factor was required to generate the PML splice variant isoform IV, which is the major structural component of APBs. We found an inverse relationship between the expression of this protein and levels of PML isoform IV mRNA. These findings suggest a novel role for ZEB1 in promoting ALT both transcriptionally and post-transcriptionally at multiple levels.

Maintaining telomere integrity is essential for cellular survival, and reactivation of telomerase or alternative lengthening of telomeres (ALT) represents a hallmark of cancer, ensuring replicative immortality. Osteosarcoma (OS), a malignancy in which many tumors rely on ALT for telomere maintenance, lacks effective therapeutic strategies targeting this pathway. This study aimed to identify and characterize novel molecular regulators of ALT activity and explore their potential as therapeutic targets in OS.Methods: Immunohistochemistry was performed to evaluate the expression of phosphorylated NPM1 (pT199-NPM1) in OS tissues. Functional experiments including NPM1 knockdown and rescue assays were conducted to assess the impact of NPM1 on break-induced telomere replication (BITR) and cell viability in ALT-positive cells. Mechanistic studies involving phosphorylation analysis, ubiquitination assays, and co-immunoprecipitation were used to determine how ATR-mediated phosphorylation of NPM1 regulates POLD3 stability and its interaction with the CST complex. Pharmacological screening was performed to identify compounds that inhibit ALT activity, followed by in vitro proliferation assays and in vivo mouse xenograft experiments to evaluate therapeutic efficacy and synergy with doxorubicin.Results: We identified pT199-NPM1 as a novel, highly expressed protein factor in ALT-positive OS tissues. NPM1 depletion impaired break-induced telomere replication and significantly reduced the viability of ALT-positive cells. ATR signaling phosphorylated NPM1 at Thr199, which stabilized POLD3 by preventing its ubiquitin-mediated degradation. Recruitment and function of pT199-NPM1 at telomeric damage sites required STN1, defining a CST/pT199-NPM1/POLD3 regulatory axis essential for ALT activity. Clinically, elevated Thr199 phosphorylation correlated with poor survival in OS patients, while expression of a phosphorylation-deficient T199A mutant failed to sustain ALT telomere maintenance. Pharmacological screening identified EPZ-6438, an EZH2 inhibitor, as a potent ALT suppressor that reduced NPM1 transcription, inhibited homologous recombination-mediated telomere synthesis, and suppressed OS cell proliferation. In mouse xenografts, EPZ-6438 enhanced OS cell sensitivity to doxorubicin, suggesting therapeutic synergy.Conclusions: This study uncovers a novel CST/pT199-NPM1/POLD3 regulatory module that is critical for ALT telomere maintenance in OS. Targeting NPM1 or its downstream effectors effectively suppresses ALT activity and enhances chemotherapy response. These findings provide new mechanistic insights into telomere regulation in ALT-positive tumors and highlight the therapeutic potential of NPM1-centered pathways in OS.

Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replication-associated single-ended DSBs (seDSBs). Here, we demonstrate that microhomology-mediated end joining (MMEJ) acts directly to repair seDSBs at broken replication forks (fork-MMEJ), preferentially on the leading strands, and functions cooperatively with BIR. We also showed that while fork-MMEJ is promoted by Polθ, it operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, which is distinct from canonical MMEJ (cMMEJ) defined at replication-independent double-ended DSBs (deDSBs). ATR, activated as end resection proceeds, serves as a pivotal switch to suppress fork-MMEJ while promoting BIR. Combined inactivation of ATR and Polθ synergistically kills cancer cells under high replication stress with minimal toxicity to normal cells. Together, our study provides fundamental insights into the MMEJ mechanism and offers new strategies for cancer treatment.

Heterochromatin marked by histone H3 lysine 9 (H3K9) methylation represses transcription of pericentromeric repeats, thereby suppressing gross chromosomal rearrangements (GCRs). However, it remains unclear how transcription causes GCRs when heterochromatin is lost. Using fission yeast, we show that transcriptional Pausing–Backtracking–Restart (PBR) cycles accumulate R-loops, leading to GCRs. DNA–RNA immunoprecipitation (DRIP) revealed that loss of Clr4, the H3K9 methyltransferase, increased R-loops at pericentromeric repeats. Overexpression of RNaseH1 in clr4∆ cells reduced both R-loops and GCRs, demonstrating that R-loops cause GCRs. Tfs1/TFIIS and Ubp3, required for transcriptional restart, and Seb1, involved in pausing at pericentromeres, were required for R-loop accumulation and GCRs, implicating PBR cycles in the formation of genotoxic R-loops. We also demonstrate that Rad52 recombinase localizes to pericentromeric repeats and facilitates GCRs in clr4∆ cells. rad52–R45K, which impairs single-strand annealing (SSA), reduced GCRs. A single-stranded DNA (ssDNA) region within an R-loop may anneal to homologous ssDNA to form Annealing-induced DNA–RNA-loops (ADR-loops). Indeed, Rad52 facilitated ADR-loop formation in vitro. Polδ was also involved in GCRs. These data suggest that, when heterochromatin is lost, transcriptional PBR cycles accumulate R-loops at pericentromeric repeats, and Rad52-dependent SSA converts R-loops into ADR-loops followed by Polδ-dependent break-induced replication (BIR), resulting in homology-mediated GCRs.

Break-induced replication (BIR) is a primary homologous recombination pathway for repairing one-ended double-strand DNA breaks, including those arising from collapsed replication forks and eroded telomeres. BIR frequently leads to loss of heterozygosity, genetic mutations, and gross chromosomal rearrangements, all hallmarks of cancer. Here, we conducted a genome-wide screen that allowed us to identify and validate the involvement of 33 novel yeast genes in BIR. We report that, while DNA damage and spindle checkpoint machineries are both required to delay nuclear division and provide adequate time for BIR to complete, the spindle position checkpoint is required to coordinate between nuclear division and cytokinesis. Furthermore, we show that two nucleopore proteins play a sequential role during BIR: Nup84 acts before DNA synthesis, while Nup188 functions later to support repair completion. Given the conservation of BIR between yeast and humans and the role of BIR in cancer development, human homologs of the identified BIR proteins may represent promising targets for anti-cancer therapeutics.

Double-strand breaks (DSBs) pose a significant threat to chromosome stability and, if left unrepaired, can result in chromosome rearrangements. Canonical DNA repair pathways mitigate these risks. However, if these repair mechanisms fail to repair the DSB, alternative repair pathways, such as break-induced replication, single-strand annealing, and de novo telomere addition (dnTA), can be utilized. Yeast subtelomeric regions are hotspots of recombination, while interstitial telomere-like sites can promote dnTA. In yeast, dnTA sites, termed SiRTAs (sites of repair-associated telomere addition), require Cdc13 association. We identified the ubiquitin protease Ubp10 as a positive regulator of dnTA at SiRTAs. Loss of UBP10 reduces dnTA frequency but increases the frequency of other chromosomal rearrangements at SiRTAs. SiRTAs utilize the repetitive subtelomeric regions of donor chromosomes to facilitate rearrangements, with a fraction occurring independently of RAD51 and requiring Sir4 and Sir2 components of the SIR complex. A DNA sequence capable of binding Cdc13 is necessary and sufficient to stimulate translocations in the absence of UBP10. This study highlights the diversity of DNA repair mechanisms at SiRTAs, advancing our understanding of telomere maintenance and chromosomal rearrangement formation.

PurposeRing chromosomes (RCs) are rare cytogenetic abnormalities involving copy-number variants and chromosome instability. Identifying the breakage-fusion sequences of RCs at nucleotide-level resolution can elucidate the cytogenomic rearrangements and ring formation mechanisms.MethodsThis study used short-read genomic sequencing (srGS) and long-read genomic sequencing (lrGS) alongside the telomere-to-telomere reference genome to characterize the breakage-fusion events of 17 RC cases.ResultsComplete RCs without loss of euchromatin by a fusion of subtelomeric or telomeric regions were noted in a RC14 and a RC20. Incomplete RCs with intrachromosomal copy-number variants were noted in 15 cases, including a RC3, a RC4, 4 RC13s, a RC14, 3 RC18s, a RC21, 3 RC22s, and an RCY. srGS defined breakage-fusion sequences in single-copy sequences, and lrGS mapped subtelomeric and pericentric repetitive sequences using the telomere-to-telomere reference genome. The breakage-fusion sequences revealed ring formation mechanisms by intrastrand nonhomology end joining in 5 RCs, microhomology-mediated end joining in 8 RCs, and microhomology-mediated break-induced replication in 4 RCs.ConclusionThis study demonstrated the analytic validity and diagnostic utility of srGS and lrGS in delineating the genomic rearrangements in RCs for better interpreting clinico-cytogenomic correlations and further analysis of RC behavior in cell cycles.

SUMMARYThe primary role of break-induced replication (BIR) is to repair single-ended double-strand breaks (seDSBs) generated at broken replication forks and eroding telomeres. In this study, we demonstrated that when senataxin (SETX), an RNA/DNA helicase, is defective, hyper-recombination using the BIR mechanism is induced at R-loops/hybrid-accumulated double-ended DSBs (deDSBs), uncovering a role for BIR in the repair of R-loops/hybrids-associated deDSBs. Intriguingly, the loss of SETX not only triggers non-canonical hyper-end resection requiring RAD52 and XPF but also stalls Polα-primase-initiated end-fill DNA synthesis due to the accumulation of RNA/DNA hybrids on single-strand DNA (ssDNA) overhangs at deDSBs. This conflict between fill-in DNA synthesis and accumulated hybrids induces PCNA ubiquitination and PIF1 loading, thereby initiating the BIR mechanism at deDSBs. Hyper-resection further enhances PCNA ubiquitination and PIF1 loading, driving BIR-mediated hyper-recombination. Moreover, dysfunctional SETX is synthetic lethal with loss of PIF1, RAD52, or XPF, offering new strategies for targeted treatment of SETX-deficient tumors.