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1.
Cell Rep ; 43(4): 114116, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38625790

RESUMO

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.


Assuntos
Ciclina E , Instabilidade Genômica , Mitose , Proteínas Oncogênicas , Proteína Rad52 de Recombinação e Reparo de DNA , Humanos , Ciclina E/metabolismo , Ciclina E/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/genética , Replicação do DNA , Linhagem Celular Tumoral , Dano ao DNA , DNA/metabolismo , DNA/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia
2.
Genome Biol ; 25(1): 105, 2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38649976

RESUMO

BACKGROUND: The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged. Recently, Ki-67 has been shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, how the different roles for Ki-67 across the cell cycle are regulated and coordinated remain poorly understood. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein, coupled to its abundance and fluctuation during the cell cycle. RESULTS: Here, we use a doxycycline-inducible E3 ligase together with an auxin-inducible degron tag to achieve a rapid, acute and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allows us to show that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependent on HUWE1 ligase. This leads to defects in replication and sister chromatids cohesion, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested. CONCLUSIONS: We unveil a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.


Assuntos
Replicação do DNA , Instabilidade Genômica , Antígeno Ki-67 , Humanos , Antígeno Ki-67/metabolismo , Células HCT116 , Dano ao DNA , Ubiquitina-Proteína Ligases/metabolismo
3.
Syst Biol Reprod Med ; 70(1): 101-112, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38630598

RESUMO

MDC1 (Mediator of DNA damage Checkpoint protein 1) functions to facilitate the localization of numerous DNA damage response (DDR) components to DNA double-strand break sites. MDC1 is an integral component in preserving genomic stability and appropriate DDR regulation. There haven't been systematic investigations of MDC1 mutations that induce cancer and genomic instability. Variations in nsSNPs have the potential to modify the protein chemistry and their function. Describing functional SNPs in disease-associated genes presents a significant conundrum for investigators, it is possible to assess potential functional SNPs before conducting larger population examinations. Multiple sequences and structure-based bioinformatics strategies were implemented in the current in-silico investigation to discern potential nsSNPs of the MDC1 genes. The nsSNPs were identified with SIFT, SNAP2, Align GVGD, PolyPhen-2, and PANTHER, and their stability was determined with MUpro. The conservation, solvent accessibility, and structural effects of the mutations were identified with ConSurf, NetSurfP-2.0, and SAAFEC-SEQ respectively. Cancer-related analysis of the nsSNPs was conducted using cBioPortal and TCGA web servers. The present study appraised five nsSNPs (P1426T, P69S, P194R, P203L, and H131Y) as probably mutilating due to their existence in highly conserved regions and propensity to deplete protein stability. The nsSNPs P194R, P203L, and H131Y were concluded as deleterious and possibly damaging from the 5 prediction tools. The functional nsSNP P194R mutation is associated with skin cutaneous melanoma while no significant records were found for other nsSNPs. The present study concludes that the highly deleterious P194R mutations can potentially induce genomic instability and contribute to various cancers' pathogenesis. Developing drugs targeting these mutations can undoubtedly be advantageous in large population-based studies, particularly in the development of precision medicine.


Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Polimorfismo de Nucleotídeo Único , Mutação , Biologia Computacional , Instabilidade Genômica , Proteínas de Ciclo Celular , Proteínas Adaptadoras de Transdução de Sinal
4.
Nat Aging ; 4(4): 510-526, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38632351

RESUMO

DNA damage contributes to the aging of hematopoietic stem cells (HSCs), yet the underlying molecular mechanisms are not fully understood. In this study, we identified a heterogeneous functional role of microcephalin (MCPH1) in the nucleus and cytoplasm of mouse HSCs. In the nucleus, MCPH1 maintains genomic stability, whereas in the cytoplasm, it prevents necroptosis by binding with p-RIPK3. Aging triggers MCPH1 translocation from cytosol to nucleus, reducing its cytoplasmic retention and leading to the activation of necroptosis and deterioration of HSC function. Mechanistically, we found that KAT7-mediated lysine acetylation within the NLS motif of MCPH1 in response to DNA damage facilitates its nuclear translocation. Targeted mutation of these lysines inhibits MCPH1 translocation and, consequently, compromises necroptosis. The dysfunction of necroptosis signaling, in turn, improves the function of aged HSCs. In summary, our findings demonstrate that DNA damage-induced redistribution of MCPH1 promotes HSC aging and could have broader implications for aging and aging-related diseases.


Assuntos
Dano ao DNA , Necroptose , Camundongos , Animais , Dano ao DNA/genética , Instabilidade Genômica , Translocação Genética , Envelhecimento/genética , Células-Tronco Hematopoéticas/fisiologia
5.
Sci Rep ; 14(1): 7706, 2024 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-38565642

RESUMO

The telomere repetitive TTAGGG motif at the ends of chromosomes, serves to preserve genomic integrity and chromosomal stability. In turn, genomic instability is a hallmark of cancer-implicating telomere disturbance. Prostate cancer (PCa) shows significant ancestral disparities, with men of African ancestry at the greatest risk for aggressive disease and associated genomic instability. Yet, no study has explored the role of telomere length (TL) with respect to ancestrally driven PCa health disparities. Patient- and technically-matched tumour-blood whole genome sequencing data for 179 ancestrally defined treatment naïve PCa patients (117 African, 62 European), we assessed for TL (blood and tumour) associations. We found shortened tumour TL to be associated with aggressive PCa presentation and elevated genomic instabilities, including percentage of genome alteration and copy number gains, in men of African ancestry. For European patients, tumour TL showed significant associations with PCa driver genes PTEN, TP53, MSH2, SETBP1 and DDX11L1, while shorter blood TL (< 3200 base pairs) and tumour TL (< 2861 base pairs) were correlated with higher risk for biochemical recurrence. Concurring with previous studies linking TL to PCa diagnosis and/or prognosis, for the first time we correlated TL differences with patient ancestry with important implications for future treatments targeting telomere dysfunction.


Assuntos
Instabilidade Genômica , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Telômero/genética , Telômero/patologia , Iniquidades em Saúde
6.
Biochem J ; 481(7): 515-545, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38572758

RESUMO

Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.


Assuntos
Peptídeo Hidrolases , Ubiquitina , Humanos , Ubiquitina/genética , Ubiquitina/metabolismo , Peptídeo Hidrolases/metabolismo , Ubiquitinação , Processamento de Proteína Pós-Traducional , Ubiquitinas/genética , Ubiquitinas/metabolismo , Dano ao DNA , Endopeptidases/metabolismo , Instabilidade Genômica
7.
Genome Med ; 16(1): 48, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38566223

RESUMO

BACKGROUND: Natural killer/T cell lymphoma (NKTCL) is a clinically and genetically heterogeneous disease with poor prognosis. Genome sequencing and mutation characterization provides a powerful approach for patient stratification, treatment target discovery, and etiology identification. However, previous studies mostly concentrated on base-level mutations in primary NKTCL, whereas the large-scale genomic alterations in NKTCL and the mutational landscapes in relapsed/refractory NKTCL remain largely unexplored. METHODS: Here, we assembled whole-genome sequencing and whole-exome sequencing data from 163 patients with primary or relapsed/refractory NKTCL and compared their somatic mutational landscapes at both nucleotide and structure levels. RESULTS: Our study not only confirmed previously reported common NKTCL mutational targets like STAT3, TP53, and DDX3X but also unveiled several novel high-frequency mutational targets such as PRDM9, DST, and RBMX. In terms of the overall mutational landscape, we observed striking differences between primary and relapsed/refractory NKTCL patient groups, with the latter exhibits higher levels of tumor mutation burden, copy number variants (CNVs), and structural variants (SVs), indicating a strong signal of genomic instability. Complex structural rearrangements such as chromothripsis and focal amplification are also significantly enriched in relapsed/refractory NKTCL patients, exerting a substantial impact on prognosis. Accordingly, we devised a novel molecular subtyping system (i.e., C0-C4) with distinct prognosis by integrating potential driver mutations at both nucleotide and structural levels, which further provides an informative guidance for novel treatments that target these specific driver mutations and genome instability as a whole. CONCLUSIONS: The striking differences underlying the mutational landscapes between the primary and relapsed/refractory NKTCL patients highlight the importance of genomic instability in driving the progression of NKTCL. Our newly proposed molecular subtyping system is valuable in assisting patient stratification and novel treatment design towards a better prognosis in the age of precision medicine.


Assuntos
Linfoma Extranodal de Células T-NK , Humanos , Linfoma Extranodal de Células T-NK/genética , Linfoma Extranodal de Células T-NK/patologia , Mutação , Instabilidade Genômica , Nucleotídeos , Células Matadoras Naturais , Histona-Lisina N-Metiltransferase/genética
8.
Artigo em Inglês | MEDLINE | ID: mdl-38575248

RESUMO

Type 2 diabetes mellitus (T2D) is a metabolic disease, which occurs largely due to unhealthy lifestyle. As oxidative stress is believed to promote T2D, by inducing damage to lipids, proteins, and DNA, appropriate dietary interventions seem critical to prevent, manage, and even reverse this condition. Brazil nuts (Bertholletia excelsa, H.B.K.) are nature's richest source of selenium, a mineral that has shown several health benefits. Therefore, this study aims to assess the effects of selenium consumption, through Brazil nuts, on biochemical and oxidative stress parameters, and genomic instability in T2D patients. We recruited 133 patients with T2D, registered in the Integrated Clinics of the University of Southern Santa Catarina (Brazil). Participants consumed one Brazil nut a day for six months. Blood samples and exfoliated buccal cells were collected at the beginning and the end of the intervention. The glycemic profile, lipid profile, renal profile and hepatic profile, DNA damage and selenium content were evaluated. A total of 74 participants completed the intervention. Brazil nut consumption increased selenium and GSH levels, GPx, and CAT activity while DCF and nitrites levels decreased. Total thiols increased, and protein carbonyl and MDA levels decreased. Levels of baseline and oxidative DNA damage in T2D patients were significantly decreased, as well as the frequency of micronuclei and nuclear buds. The fasting glucose levels, HDL and LDL cholesterol, and GGT levels that increased significantly in patients with type 2 diabetes were significantly reduced with nut consumption. Our results show an increase in antioxidant activity, along with reductions of protein and lipid oxidation as well as DNA damage, suggesting that Brazil nut consumption could be an ally in reducing oxidative stress and modulating the genomic instability in T2D patients.


Assuntos
Bertholletia , Diabetes Mellitus Tipo 2 , Selênio , Humanos , Bertholletia/química , Selênio/farmacologia , Sobrepeso , Diabetes Mellitus Tipo 2/genética , Mucosa Bucal , Lipídeos , Dano ao DNA , Instabilidade Genômica
9.
BMC Cancer ; 24(1): 415, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38575974

RESUMO

BACKGROUND: Genome stability is maintained by the DNA damage repair (DDR) system composed of multiple DNA repair pathways of hundreds of genes. Germline pathogenic variation (PV) in DDR genes damages function of the affected DDR genes, leading to genome instability and high risk of diseases, in particular, cancer. Knowing evolutionary origin of the PVs in human DDR genes is essential to understand the etiology of human diseases. However, answer to the issue remains largely elusive. In this study, we analyzed evolutionary origin for the PVs in human DDR genes. METHODS: We identified 169 DDR genes by referring to various databases and identified PVs in the DDR genes of modern humans from ClinVar database. We performed a phylogenetic analysis to analyze the conservation of human DDR PVs in 100 vertebrates through cross-species genomic data comparison using the phyloFit program of the PHAST package and visualized the results using the GraphPad Prism software and the ggplot module. We identified DDR PVs from over 5000 ancient humans developed a database to host the DDR PVs ( https://genemutation.fhs.um.edu.mo/dbDDR-AncientHumans ). Using the PV data, we performed a molecular archeological analysis to compare the DDR PVs between modern humans and ancient humans. We analyzed evolution selection of DDR genes across 20 vertebrates using the CodeML in PAML for phylogenetic analysis. RESULTS: Our phylogenic analysis ruled out cross-species conservation as the origin of human DDR PVs. Our archeological approach identified rich DDR PVs shared between modern and ancient humans, which were mostly dated within the last 5000 years. We also observed similar pattern of quantitative PV distribution between modern and ancient humans. We further detected a set of ATM, BRCA2 and CHEK2 PVs shared between human and Neanderthals. CONCLUSIONS: Our study reveals that human DDR PVs mostly arose in recent human history. We propose that human high cancer risk caused by DDR PVs can be a by-product of human evolution.


Assuntos
Reparo do DNA , Neoplasias , Humanos , Filogenia , Reparo do DNA/genética , Genes BRCA2 , Neoplasias/genética , Instabilidade Genômica , Dano ao DNA/genética , Predisposição Genética para Doença
10.
Proc Natl Acad Sci U S A ; 121(17): e2315926121, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38625945

RESUMO

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.


Assuntos
Candida albicans , Edição de Genes , Humanos , Candida albicans/genética , Interferência de RNA , Saccharomyces cerevisiae/metabolismo , Instabilidade Genômica
11.
Sci Rep ; 14(1): 8797, 2024 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-38627415

RESUMO

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.


Assuntos
Mieloma Múltiplo , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo do DNA , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Fatores de Transcrição
12.
J Clin Invest ; 134(7)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38557488

RESUMO

While breast cancer 2 (BRCA2) loss of heterozygosity (LOH) promotes cancer initiation, it can also induce death in nontransformed cells. In contrast, mismatch repair gene mutL homolog 1 (MLH1) is a tumor-suppressor gene that protects cells from cancer development through repairing mismatched base pairs during DNA mismatch repair (MMR). Sengodan et al., in this issue of the JCI, reveal an interplay between the 2 genes: MLH1 promoted the survival of BRCA2-deficient cells independently of its MMR function. MLH1 protected replication forks from degradation, while also resolving R-loops, thereby reducing genomic instability. Moreover, MLH1 expression was regulated directly by estrogen, shedding light into the hormone-responsive nature of many BRCA2 mutant breast cancers. These results provide important insight into the genetics that drive the initiation of BRCA2-mutated breast cancers.


Assuntos
Neoplasias da Mama , Proteína 1 Homóloga a MutL , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Instabilidade Genômica , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo
13.
Nat Commun ; 15(1): 2132, 2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38459011

RESUMO

Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adapter for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS-STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination.


Assuntos
Replicação do DNA , Neoplasias , Animais , Humanos , Camundongos , DNA , Instabilidade Genômica , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Imunidade Inata , Proteína Homóloga a MRE11/metabolismo , Neoplasias/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
14.
Cell Mol Life Sci ; 81(1): 123, 2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38459149

RESUMO

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.


Assuntos
Células-Tronco Neurais , Reparo de DNA por Recombinação , Animais , Camundongos , Arginina/metabolismo , Reparo do DNA , Instabilidade Genômica , Genômica , Histonas/genética , Histonas/metabolismo , Células-Tronco Neurais/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo
15.
Methods Mol Biol ; 2760: 371-392, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468099

RESUMO

Genetic engineering has revolutionized our ability to manipulate DNA and engineer organisms for various applications. However, this approach can lead to genomic instability, which can result in unwanted effects such as toxicity, mutagenesis, and reduced productivity. To overcome these challenges, smart design of synthetic DNA has emerged as a promising solution. By taking into consideration the intricate relationships between gene expression and cellular metabolism, researchers can design synthetic constructs that minimize metabolic stress on the host cell, reduce mutagenesis, and increase protein yield. In this chapter, we summarize the main challenges of genomic instability in genetic engineering and address the dangers of unknowingly incorporating genomically unstable sequences in synthetic DNA. We also demonstrate the instability of those sequences by the fact that they are selected against conserved sequences in nature. We highlight the benefits of using ESO, a tool for the rational design of DNA for avoiding genetically unstable sequences, and also summarize the main principles and working parameters of the software that allow maximizing its benefits and impact.


Assuntos
Engenharia Genética , Instabilidade Genômica , Humanos , DNA/genética , Proteínas/genética
16.
Nature ; 628(8006): 145-153, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38538785

RESUMO

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.


Assuntos
Região CA1 Hipocampal , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Inflamação , Memória , Receptor Toll-Like 9 , Animais , Feminino , Masculino , Camundongos , Envelhecimento/genética , Envelhecimento/patologia , Região CA1 Hipocampal/fisiologia , Centrossomo/metabolismo , Disfunção Cognitiva/genética , Condicionamento Clássico , Matriz Extracelular/metabolismo , Medo , Instabilidade Genômica/genética , Histonas/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Memória/fisiologia , Transtornos Mentais/genética , Doenças Neurodegenerativas/genética , Doenças Neuroinflamatórias/genética , Neurônios/metabolismo , Neurônios/patologia , Membrana Nuclear/patologia , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismo
17.
Nucleic Acids Res ; 52(7): 3493-3509, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38442257

RESUMO

Gene-strand bias is a characteristic feature of bacterial genome organization wherein genes are preferentially encoded on the leading strand of replication, promoting co-orientation of replication and transcription. This co-orientation bias has evolved to protect gene essentiality, expression, and genomic stability from the harmful effects of head-on replication-transcription collisions. However, the origin, variation, and maintenance of gene-strand bias remain elusive. Here, we reveal that the frequency of inversions that alter gene orientation exhibits large variation across bacterial populations and negatively correlates with gene-strand bias. The density, distance, and distribution of inverted repeats show a similar negative relationship with gene-strand bias explaining the heterogeneity in inversions. Importantly, these observations are broadly evident across the entire bacterial kingdom uncovering inversions and inverted repeats as primary factors underlying the variation in gene-strand bias and its maintenance. The distinct catalytic subunits of replicative DNA polymerase have co-evolved with gene-strand bias, suggesting a close link between replication and the origin of gene-strand bias. Congruently, inversion frequencies and inverted repeats vary among bacteria with different DNA polymerases. In summary, we propose that the nature of replication determines the fitness cost of replication-transcription collisions, establishing a selection gradient on gene-strand bias by fine-tuning DNA sequence repeats and, thereby, gene inversions.


Assuntos
Bactérias , Replicação do DNA , Evolução Molecular , Genoma Bacteriano , Replicação do DNA/genética , Bactérias/genética , Bactérias/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Sequências Repetidas Invertidas , Origem de Replicação/genética , Transcrição Gênica , Instabilidade Genômica
18.
Science ; 383(6690): 1441-1448, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38547292

RESUMO

Mitotic duration is tightly constrained, and extended mitosis is characteristic of problematic cells prone to chromosome missegregation and genomic instability. We show here that mitotic extension leads to the formation of p53-binding protein 1 (53BP1)-ubiquitin-specific protease 28 (USP28)-p53 protein complexes that are transmitted to, and stably retained by, daughter cells. Complexes assembled through a Polo-like kinase 1-dependent mechanism during extended mitosis and elicited a p53 response in G1 that prevented the proliferation of the progeny of cells that experienced an approximately threefold extended mitosis or successive less extended mitoses. The ability to monitor mitotic extension was lost in p53-mutant cancers and some p53-wild-type (p53-WT) cancers, consistent with classification of TP53BP1 and USP28 as tumor suppressors. Cancers retaining the ability to monitor mitotic extension exhibited sensitivity to antimitotic agents.


Assuntos
Proliferação de Células , Mitose , Neoplasias , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina Tiolesterase , Humanos , Proliferação de Células/genética , Instabilidade Genômica , Mitose/efeitos dos fármacos , Mitose/genética , Neoplasias/genética , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , 60688/metabolismo , Antimitóticos/farmacologia , Resistencia a Medicamentos Antineoplásicos
19.
DNA Repair (Amst) ; 137: 103669, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38507953

RESUMO

The SMC5/6 complex is evolutionarily conserved across all eukaryotes and plays a pivotal role in preserving genomic stability. Mutations in genes encoding SMC5/6 complex subunits have been associated with human lung disease, immunodeficiency, and chromosome breakage syndrome. Despite its critical importance, much about the SMC5/6 complex remains to be elucidated. Various evidences have suggested possible role of a subunit of the SMC5/6 complex, NSE1, in chromosome segregation and DNA repair. Current knowledge regarding the role of NSE1 is primarily derived from single-cell-based analyses in yeasts, Arabidopsis thaliana, and human cell lines. However, our understanding of its function is still limited and requires further investigation. This study delves into the role of nse-1 in Caenorhabditis elegans, revealing its involvement in meiotic recombination and DNA repair. nse-1 mutants display reduced fertility, increased male incidence, and increased sensitivity to genotoxic chemicals due to defects in meiotic chromosome segregation and DNA repair. These defects manifest as increased accumulation of RAD-51 foci, increased chromosome fragmentation, and susceptibility to MMS, cisplatin, and HU. Furthermore, nse-1 mutation exacerbates germ cell death by upregulating ced-13 and egl-1 genes involved in the CEP-1/p53-mediated apoptotic pathway. NSE-1 is essential for the proper localization of NSE-4 and MAGE-1 on the chromosomes. Collectively, these findings firmly establish nse-1 as a crucial factor in maintaining genomic stability.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Masculino , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Meiose , Instabilidade Genômica , Proteínas de Caenorhabditis elegans/genética
20.
Biochim Biophys Acta Mol Basis Dis ; 1870(4): 167116, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38447882

RESUMO

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.


Assuntos
Aurora Quinase A , Proteínas de Ciclo Celular , Humanos , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína Supressora de Tumor p53/genética , Segregação de Cromossomos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Instabilidade Genômica , Instabilidade Cromossômica/genética , Cromossomos/metabolismo
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