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A substantial number of human cancers are telomerase-negative and elongate physiologically damaged telomeres through a break-induced replication (BIR)-based mechanism known as alternative lengthening of telomeres (ALT). We recently demonstrated that inhibiting the transcription of the telomeric long noncoding RNA TERRA suppresses telomere damage and ALT features, indicating that telomere transcription is a main trigger of ALT activity. Here we show that experimentally increased TERRA transcription not only increases ALT features, as expected, but also causes rapid loss of telomeric DNA through a pathway that requires the endonuclease Mus81. Our data indicate that the ALT mechanism can endanger telomere integrity if not properly controlled and point to TERRA transcription as a uniquely versatile target for therapy.

Meiotic crossovers/chiasmata are not randomly distributed and strictly controlled. The mechanisms behind crossover (CO) patterning remain largely unknown. In Allium cepa, as in the vast majority of plants and animals, COs predominantly occur in the distal 2/3 of the chromosome arm, while in Allium fistulosum they are strictly localized in the proximal region. We investigated the factors that may contribute to the pattern of COs in A. cepa, A. fistulosum and their F1 diploid (2n = 2x = 8C + 8F) and F1 triploid (2n = 3x = 16F + 8C) hybrids. The genome structure of F1 hybrids was confirmed using genomic in situ hybridization (GISH). The analysis of bivalents in the pollen mother cells (PMCs) of the F1 triploid hybrid showed a significant shift in the localization of COs to the distal and interstitial regions. In F1 diploid hybrid, the COs localization was predominantly the same as that of the A. cepa parent. We found no differences in the assembly and disassembly of ASY1 and ZYP1 in PMCs between A. cepa and A. fistulosum, while F1 diploid hybrid showed a delay in chromosome pairing and a partial absence of synapsis in paired chromosomes. Immunolabeling of MLH1 (class I COs) and MUS81 (class II COs) proteins showed a significant difference in the class I/II CO ratio between A. fistulosum (50%:50%) and A. cepa (73%:27%). The MLH1:MUS81 ratio at the homeologous synapsis of F1 diploid hybrid (70%:30%) was the most similar to that of the A. cepa parent. F1 triploid hybrid at the A. fistulosum homologous synapsis showed a significant increase in MLH1:MUS81 ratio (60%:40%) compared to the A. fistulosum parent. The results suggest possible genetic control of CO localization. Other factors affecting the distribution of COs are discussed.

The eukaryotic cell cycle is conventionally viewed as comprising several discrete steps, each of which must be completed before the next one is initiated. However, emerging evidence suggests that incompletely replicated, or unresolved, chromosomes from S‐phase can persist into mitosis, where they present a potential threat to the faithful segregation of sister chromatids. In this review, we provide an overview of the different classes of loci where this ‘unfinished S‐phase business’ can lead to a variety of cytogenetically distinct DNA structures throughout the various steps of mitosis. Furthermore, we discuss the potential ways in which cells might not only tolerate this inevitable aspect of chromosome biology, but also exploit it to assist in the maintenance of genome stability. Hickson and colleagues review emerging concepts on how replication problems are frequently processed only in subsequent cell‐cycle stages, and postulate that cells may even exploit this to ensure genome stability maintenance during cell division.

Maintaining genome integrity is essential, yet how DNA repair is balanced across life stages remains poorly understood. Here we uncover an epitranscriptomic mechanism in the fungal pathogen Fusarium graminearum that alleviates a trade-off between heat-stress adaptation and sexual reproduction. We show that FgMus81 acts independently of its nuclease activity and canonical partner FgMms4, and has dosage-dependent, stage-specific functions: restrained levels support meiosis, whereas elevated levels promote heat-stress survival. We identify a sexual stage-specific A-to-I RNA editing event that recodes FgMus81 (N420D) and tunes its abundance to meet meiotic demands without compromising stress resilience. Notably, both pre-editing and post-editing isoforms support meiotic interhomolog crossovers, but the post-editing isoform impairs mitotic recombination. Conservation of this editing across Sordariomycetes suggests evolutionary selection for stage-specific control. Together, these findings reveal an epitranscriptomic switch that partitions Mus81 functions across life stages and identify adaptive RNA editing as a regulator of homologous recombination in fungal pathogens. Maintaining genome integrity during different life stages is critical. Here, the authors reveal an RNA editing mechanism in the fungal pathogen Fusarium graminearum that adjusts Mus81 protein function to balance heat-stress survival and sexual reproduction. This stage-specific editing fine-tunes DNA repair for adaptation and reproduction.

RecQ helicases are involved in the processing of DNA structures arising during replication, recombination, and repair throughout all kingdoms of life. Mutations of different RecQ homologues are responsible for severe human diseases, such as Blooms (BLM) or Werner (WRN) syndrome. The loss of RecQ function is often accompanied by hyperrecombination caused by a lack of crossover suppression. In the Arabidopsis genome seven different RecQ genes are present. Two of them (AtRECQ4A and 4B) arose because of a recent duplication and are still nearly 70% identical on a protein level. Knockout of these genes leads to antagonistic phenotypes: the RECQ4A mutant shows sensitivity to DNA-damaging agents, enhanced homologous recombination (HR) and lethality in a mus81 background. Moreover, mutation of RECQ4A partially suppresses the lethal phenotype of an AtTOP3α mutant, a phenomenon that had previously been demonstrated for RecQ homologues of unicellular eukaryotes only. Together, these facts strongly suggest that in plants RECQ4A is functionally equivalent to SGS1 of Saccharomyces cerevisiae and the mammalian BLM protein. In stark contrast, mutants of the closely related RECQ4B are not mutagen-sensitive, not viable in a mus81 background, and unable to suppress the induced lethality caused by loss of TOP3α. Moreover, they are strongly impaired in HR. Thus, AtRECQ4B is specifically required to promote but not to suppress crossovers, a role in which it differs from all eukaryotic RecQ homologues known.

The cellular pathways that restart stalled replication forks are essential for genome stability and tumor prevention. However, how many of these pathways exist in cells and how these pathways are selectively activated remain unclear. Here, we describe two major fork restart pathways, and demonstrate that their selection is governed by 53BP1 and BRCA1, which are known to control the pathway choice to repair double-strand DNA breaks (DSBs). Specifically, 53BP1 promotes a fork cleavage-free pathway, whereas BRCA1 facilitates a break-induced replication (BIR) pathway coupled with SLX-MUS complex-mediated fork cleavage. The defect in the first pathway, but not DSB repair, in a 53BP1 mutant is largely corrected by disrupting BRCA1, and vice versa. Moreover, PLK1 temporally regulates the switch of these two pathways through enhancing the assembly of the SLX-MUS complex. Our results reveal two distinct fork restart pathways, which are antagonistically controlled by 53BP1 and BRCA1 in a DSB repair-independent manner.

Background Identification of genomic biomarkers to predict the anticancer effects of indicated drugs is considered a promising strategy for the development of precision medicine. DNA endonuclease MUS81 plays a pivotal role in various biological processes during malignant diseases, mainly in DNA damage repair and replication fork stability. Our previous study reported that MUS81 was highly expressed and linked to tumor metastasis in gastric cancer; however, its therapeutic value has not been fully elucidated. Methods Bioinformatics analysis was used to define MUS81-related differential genes, which were further validated in clinical tissue samples. Gain or loss of function MUS81 cell models were constructed to elucidate the effect and mechanism of MUS81 on WEE1 expression. Moreover, the antitumor effect of targeting MUS81 combined with WEE1 inhibitors was verified using in vivo and in vitro assays. Thereafter, the cGAS/STING pathway was evaluated, and the therapeutic value of MUS81 for immunotherapy of gastric cancer was determined. Results In this study, MUS81 negatively correlated with the expression of cell cycle checkpoint kinase WEE1. Furthermore, we identified that MUS81 regulated the ubiquitination of WEE1 via E-3 ligase β-TRCP in an enzymatic manner. In addition, MUS81 inhibition could sensitize the anticancer effect of the WEE1 inhibitor MK1775 in gastric cancer in vitro and in vivo. Interestingly, when MUS81 was targeted, it increased the accumulation of cytosolic DNA induced by MK1775 treatment and activated the DNA sensor STING-mediated innate immunity in the gastric cancer cells. Thus, the WEE1 inhibitor MK1775 specifically enhanced the anticancer effect of immune checkpoint blockade therapy in MUS81 deficient gastric cancer cells. Conclusions Our data provide rational evidence that targeting MUS81 could elevate the expression of WEE1 by regulating its ubiquitination and could activate the innate immune response, thereby enhancing the anticancer efficacy of WEE1 inhibitor and immune checkpoint blockade combination therapy in gastric cancer cells.

Background DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. Results This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. Conclusions Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

High-risk neuroblastoma is a pediatric tumor that originates from immature sympathetic neuroblasts and is characterized by a low mutational burden. Nevertheless, it frequently presents with highly recurrent chromosomal imbalances, including MYCN amplification and gain of chromosome 17q. Highly proliferative cancers, such as neuroblastoma, exhibit significant DNA replication stress, rendering tumor cells dependent on ATR-CHK1 signalling and DNA damage repair pathways. We previously hypothesized that gene dosage effects resulting from 17q copy number alterations of non-mutated genes involved in replication stress resistance could offer novel therapeutic opportunities. To identify critical candidate genes and pathways driving high-risk neuroblastoma, we performed an integrated bioinformatics analysis, and identified the BRIP1 gene, encoding the FANCJ protein, as top-ranked 17q candidate. FANCJ is involved in multiple processes that alleviate replication stress. In the absence of specific FANCJ-targeting compounds, we evaluated the phenotypic and molecular effects of pharmacological inhibition of the MUS81 endonuclease, which functions downstream of FANCJ in restarting stalled replication forks. When combined with CHK1 inhibition, we observed synergistic effects on neuroblastoma cell growth and survival, supporting further development of on-target MUS81 inhibitors for in vivo preclinical testing and future clinical trials aimed at overcoming replication stress resistance in high-risk neuroblastoma.

This project attempts to clarify the expression of MUS81 in castration-resistant prostate cancer (CRPC) and the effect on drug sensitivity to Olaparib. We collected clinical surgical samples of patients who were suffering from benign prostatic hyperplasia (BPH), common prostate cancer (PCa), and castration-resistant prostate cancer (CRPC) and detected the expression of MUS81 in healthy prostate epithelial cells, PCa cells, and androgen-independent PCa cells. We subsequently performed CCK-8 assays, flow cytometry, and Transwell invasion and migration assay to determine the proliferation, apoptosis, invasion, and metastasis abilities of transfected CRPC cells as well as drug toxicity of Olaparib to CRPC cells. The expression of MUS81 indicated marked upregulation in PCa and CRPC tissues, compared with the level of MUS81 in BPH tissues. MUS81 silencing inhibited the proliferation of CRPC cells and promoted their sensitivity to Olaparib. MUS81 silencing in CRPC cells remarkably accelerated cell apoptosis and greatly inhibited cell invasion and metastasis after Olaparib administration. MUS81 silencing in CRPC cells has significantly enhanced the sensitivity of cells to Olaparib, which provides evidence for the prediction of Olaparib resistance in CRPC cells by the MUS81 gene and is expected to become a promising gene target in CRPC therapy.