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Genes & Development Apr 2024The post-translational modification of proteins by SUMO is crucial for cellular viability and mammalian development in part due to the contribution of SUMOylation to...
The post-translational modification of proteins by SUMO is crucial for cellular viability and mammalian development in part due to the contribution of SUMOylation to genome duplication and repair. To investigate the mechanisms underpinning the essential function of SUMO, we undertook a genome-scale CRISPR/Cas9 screen probing the response to SUMOylation inhibition. This effort identified 130 genes whose disruption reduces or enhances the toxicity of TAK-981, a clinical-stage inhibitor of the SUMO E1-activating enzyme. Among the strongest hits, we validated and characterized NFATC2IP, an evolutionarily conserved protein related to the fungal Esc2 and Rad60 proteins that harbors tandem SUMO-like domains. Cells lacking NFATC2IP are viable but are hypersensitive to SUMO E1 inhibition, likely due to the accumulation of mitotic chromosome bridges and micronuclei. NFATC2IP primarily acts in interphase and associates with nascent DNA, suggesting a role in the postreplicative resolution of replication or recombination intermediates. Mechanistically, NFATC2IP interacts with the SMC5/6 complex and UBC9, the SUMO E2, via its first and second SUMO-like domains, respectively. AlphaFold-Multimer modeling suggests that NFATC2IP positions and activates the UBC9-NSMCE2 complex, the SUMO E3 ligase associated with SMC5/SMC6. We conclude that NFATC2IP is a key mediator of SUMO-dependent genomic integrity that collaborates with the SMC5/6 complex.
Topics: Cell Cycle Proteins; DNA Damage; Sumoylation; Ubiquitin-Protein Ligases; Humans; Genomic Instability
PubMed: 38503515
DOI: 10.1101/gad.350914.123 -
Nature May 2024RAD52 is important for the repair of DNA double-stranded breaks, mitotic DNA synthesis and alternative telomere length maintenance. Central to these functions, RAD52...
RAD52 is important for the repair of DNA double-stranded breaks, mitotic DNA synthesis and alternative telomere length maintenance. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA) and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal, and aberrant expression of RAD52 is associated with poor cancer prognosis. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.
Topics: Humans; Cryoelectron Microscopy; DNA, Single-Stranded; Models, Molecular; Protein Binding; Rad52 DNA Repair and Recombination Protein; Replication Protein A; Multiprotein Complexes; Protein Domains; Binding Sites
PubMed: 38658755
DOI: 10.1038/s41586-024-07347-7 -
Nature Apr 2024RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs). However, the mechanism by which...
RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs). However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.
Topics: Humans; Cryoelectron Microscopy; DNA; DNA Breaks, Double-Stranded; DNA Repair; Nucleosomes; Protein Subunits; Rad51 Recombinase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Mutation; Protein Domains; Histones; Protein Binding
PubMed: 38509361
DOI: 10.1038/s41586-024-07196-4 -
Molecular Therapy. Nucleic Acids Sep 2023Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and...
Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and confer long-term transgene expression. Despite an ever-growing library of capsid variants, widespread delivery of AAVs in the adult central nervous system remains a challenge. We have previously demonstrated successful distribution of secreted proteins by infection of the ependyma, a layer of post-mitotic epithelial cells lining the ventricles of the brain and central column of the spinal cord, and subsequent protein delivery via the cerebrospinal fluid (CSF). Here we define a functional ependyma promoter to enhance expression from this cell type. Using RNA sequencing on human autopsy samples, we identified disease- and age-independent ependyma gene signatures. Associated promoters were cloned and screened as libraries in mouse and rhesus macaque to reveal cross-species function of a human DNA-derived von Willebrand factor domain containing 3A () promoter. When tested in mice, our promoter drove strong, ependyma-localized expression of eGFP and increased secreted ApoE protein levels in the CSF by 2-12× over the ubiquitous iCAG promoter.
PubMed: 37547292
DOI: 10.1016/j.omtn.2023.07.016 -
Molecular Cell Apr 2024Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining...
Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.
Topics: Animals; Chromatin; Nuclear Proteins; Transcription Factors; DNA; DNA End-Joining Repair; Histones; Chromosome Pairing; Ku Autoantigen; Mammals
PubMed: 38423014
DOI: 10.1016/j.molcel.2024.02.002 -
The EMBO Journal Jun 2024MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the...
MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that disabling MCM8 in mice resulted in proliferation defects of primordial germ cells (PGCs) and ultimately impaired fertility. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results show MCM8 interacts with R-loop-resolving factors to prevent R-loop-induced DNA damage, which may contribute to the maintenance of genome integrity of PGCs and reproductive reserve establishment. Our findings thus reveal an essential role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells.
PubMed: 38858601
DOI: 10.1038/s44318-024-00134-0 -
Cell Reports Apr 2024Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly...
Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly rely on DNA repair, including RAD52-mediated break-induced replication during interphase. We show that not all DNA lesions induced by Cyclin E1 overexpression are resolved during interphase. While DNA lesions upon Cyclin E1 overexpression are induced in S phase, a significant fraction of these lesions is transmitted into mitosis. Cyclin E1 overexpression triggers mitotic DNA synthesis (MiDAS) in a RAD52-dependent fashion. Chemical or genetic inactivation of MiDAS enhances mitotic aberrations and persistent DNA damage. Mitosis-specific degradation of RAD52 prevents Cyclin E1-induced MiDAS and reduces the viability of Cyclin E1-overexpressing cells, underscoring the relevance of RAD52 during mitosis to maintain genomic integrity. Finally, analysis of breast cancer samples reveals a positive correlation between Cyclin E1 amplification and RAD52 expression. These findings demonstrate the importance of suppressing mitotic defects in Cyclin E1-overexpressing cells through RAD52.
Topics: Humans; Cyclin E; Genomic Instability; Rad52 DNA Repair and Recombination Protein; Mitosis; Oncogene Proteins; DNA Replication; Cell Line, Tumor; DNA Damage; DNA; Breast Neoplasms
PubMed: 38625790
DOI: 10.1016/j.celrep.2024.114116 -
Journal of Ovarian Research Aug 2023Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance mice detected meiotic arrest of...
Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance mice detected meiotic arrest of primordial germ cells (PGCs) as early as embryonic day (E) 13.5 and exhibited decreased ovarian reserve after birth. However, the mechanism of Fance defect leading to dysgenesis of PGCs is unclear. We aimed to explore the effect of Fance defects on mitotic proliferation of PGCs. Combined with transcriptomic sequencing and validation, we examined the effect of Fance defects on cell cycle, transcription-replication conflicts (TRCs), and multiple DNA repair pathways in PGCs during active DNA replication at E11.5 and E12.5. Results showed Fance defects cause decreased numbers of PGCs during rapid mitosis at E11.5 and E12.5. Mitotic cell cycle progression of Fance PGCs was blocked at E11.5 and E12.5, shown by decreased cell proportions in S and G2 phases and increased cell proportions in M phase. RNA-seq suggested the mechanisms involved in DNA replication and repair. We found Fance PGCs accumulate TRCs during active DNA replication at E11.5 and E12.5. Fance PGCs down-regulate multiple DNA repair pathways at E11.5 and E12.5 including the FA pathway, homologous recombination (HR) pathway, and base excision repair (BER) pathway. In conclusion, Fance defect impaired the mitotic proliferation of PGCs leading to rapidly decreased numbers and abnormal cell cycle distribution. Proliferation inhibition of Fance PGCs was associated with accumulated TRCs and down-regulation of FA, HR, BER pathways. These provided a theoretical basis for identifying the inherited etiology and guiding potential fertility management for POI.
Topics: Animals; Mice; Cell Cycle; Cell Division; DNA Repair; Fanconi Anemia; Germ Cells; Mice, Knockout; Fanconi Anemia Complementation Group E Protein
PubMed: 37563658
DOI: 10.1186/s13048-023-01252-9 -
Science Advances Mar 2024Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of...
Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G/G-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.
Topics: Animals; Mice; Haploidy; Diploidy; Meiosis; Cell Nucleus; Chromatids
PubMed: 38457500
DOI: 10.1126/sciadv.adk9001 -
Cancer Diagnosis & Prognosis 2024Ewing sarcoma is an aggressive mesenchymal malignancy commonly affecting children and young adolescents. The molecular basis of this neoplasia is well reported with the... (Review)
Review
BACKGROUND/AIM
Ewing sarcoma is an aggressive mesenchymal malignancy commonly affecting children and young adolescents. The molecular basis of this neoplasia is well reported with the formation of the EWSR1/FLI1 fusion gene being the most common genetic finding. However, this fusion gene has not been targeted therapeutically nor is being used as a prognostic marker. Its relevance regarding the molecular steps leading to Ewing sarcoma genesis are yet to be defined. The generation of the oncogenic EWSR1/FLI1 fusion gene, can be attributed to the simultaneous introduction of two DNA double-strand breaks (DSBs). The scope of this study is to detect any association between DNA repair deficiency and the clinicopathological aspects of Ewing's sarcoma disease.
PATIENTS AND METHODS
We have conducted an expression analysis of 35 patients diagnosed with Ewing sarcoma concerning the genes involved in non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways. We have analyzed the expression levels of 6 genes involved in NHEJ (XRCC4, XRCC5, XRCC6, POLλ, POLμ) and 9 genes involved in HR (RAD51, RAD52, RAD54, BRCA1, BRCA2, FANCC, FANCD, DNTM1, BRIT1) using real time PCR. Age, sex, location of primary tumor, tumor size, KI67, mitotic count, invasion of adjacent tissues and treatment were the clinicopathological parameters included in the statistical analysis.
RESULTS
Our results show that both these DNA repair pathways are deregulated in Ewing sarcoma. In addition, low expression of the xrcc4 gene has been associated with better overall survival probability (p=0.032).
CONCLUSION
Our results, even though retrospective and in a small number of patients, highlight the importance of DSBs repair and propose a potential therapeutic target for this type of sarcoma.
PubMed: 38707718
DOI: 10.21873/cdp.10313