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1.
Nucleic Acids Res ; 50(5): 2667-2680, 2022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35166826

RESUMO

The tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSß (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSß and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA , Endonucleases , Proteína 2 Homóloga a MutS , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteína 2 Homóloga a MutS/metabolismo
2.
Nucleic Acids Res ; 49(12): e69, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-33836085

RESUMO

The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.


Assuntos
Replicação do DNA , Genômica/métodos , Animais , Linhagem Celular , Galinhas , DNA , Análise de Sequência de DNA , Transcrição Gênica
3.
Nat Struct Mol Biol ; 26(1): 58-66, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30598553

RESUMO

Common fragile sites (CFSs) are loci that are hypersensitive to replication stress and hotspots for chromosomal rearrangements in cancers. CFSs replicate late in S phase, are cell-type specific and nest in large genes. The relative impact of transcription-replication conflicts versus a low density in initiation events on fragility is currently debated. Here we addressed the relationships between transcription, replication, and instability by manipulating the transcription of endogenous large genes in chicken and human cells. We found that inducing low transcription with a weak promoter destabilized large genes, whereas stimulating their transcription with strong promoters alleviated instability. Notably, strong promoters triggered a switch to an earlier replication timing, supporting a model in which high transcription levels give cells more time to complete replication before mitosis. Transcription could therefore contribute to maintaining genome integrity, challenging the dominant view that it is exclusively a threat.


Assuntos
Instabilidade Genômica/genética , Transcrição Gênica/genética , Animais , Sítios Frágeis do Cromossomo/genética , Sítios Frágeis do Cromossomo/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Instabilidade Genômica/fisiologia , Humanos , Mitose/genética , Mitose/fisiologia
4.
Brain Res Bull ; 57(1): 11-5, 2002 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-11827732

RESUMO

The convulsant methionine sulfoximine (MSO) is a byproduct of the agenized flour commonly used for feeding domestic animals decades ago. MSO is a powerful glycogenic and epileptogenic agent, and it is an irreversible inhibitor of glutamine synthetase. This latter effect was hypothesized to be responsible for the increase in the incidence of some neuropathologies in humans, such as Alzheimer's disease or Parkinson's disease. In order to test this hypothesis, we chronically administered MSO to two inbred strains of mice, C57BL/6J and BALB/cJ, and analyzed possible alterations in learning and memory features of these mice. Mice were given 20 mg/kg of MSO three times a week for 10 weeks. Spatial learning capabilities assessed with a radial maze were not affected by the long-term MSO treatment, although activity was significantly decreased in BALB/cJ mice. Thus, our data suggest that long-term administration of non-convulsive and non-glycogenic doses of MSO do not alter the spatial memory of mice. Our results do not support the hypothesis that chronic treatment with MSO influences hippocampus-dependent learning abilities in mice.


Assuntos
Química Encefálica/efeitos dos fármacos , Glutamato-Amônia Ligase/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Deficiências da Aprendizagem/induzido quimicamente , Transtornos da Memória/induzido quimicamente , Metionina Sulfoximina/toxicidade , Doenças Neurodegenerativas/induzido quimicamente , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Química Encefálica/fisiologia , Glutamato-Amônia Ligase/metabolismo , Deficiências da Aprendizagem/metabolismo , Deficiências da Aprendizagem/fisiopatologia , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo
5.
Cell Rep ; 4(3): 420-8, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23911288

RESUMO

Cancer genomes exhibit numerous deletions, some of which inactivate tumor suppressor genes and/or correspond to unstable genomic regions, notably common fragile sites (CFSs). However, 70%-80% of recurrent deletions cataloged in tumors remain unexplained. Recent findings that CFS setting is cell-type dependent prompted us to reevaluate the contribution of CFS to cancer deletions. By combining extensive CFS molecular mapping and a comprehensive analysis of CFS features, we show that the pool of CFSs for all human cell types consists of chromosome regions with genes over 300 kb long, and different subsets of these loci are committed to fragility in different cell types. Interestingly, we find that transcription of large genes does not dictate CFS fragility. We further demonstrate that, like CFSs, cancer deletions are significantly enriched in genes over 300 kb long. We now provide evidence that over 50% of recurrent cancer deletions originate from CFSs associated with large genes.


Assuntos
Sítios Frágeis do Cromossomo , Células Epiteliais/ultraestrutura , Células Eritroides/ultraestrutura , Neoplasias/genética , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Eritroides/metabolismo , Células HCT116 , Humanos , Células K562 , Transcrição Gênica
6.
Cancer Biol Ther ; 6(5): 719-23, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17426442

RESUMO

The treatment of malignant brain gliomas remains a challenge, despite the availability of the classical triad of surgery, radiotherapy, and chemotherapy. There is thus the need for investigations into other forms of treatment strategies, such as gene therapy. Using antisense technology we have targeted glycogen metabolism, since malignant astrocytes present a high content of glycogen. In vitro rat C6­glioma cells, transfected with antisense glycogen synthase (C6­AS cells) exhibited a decreased expression of glycogen synthase and reduced activity of glycogen synthesis, along with attenuated invasiveness. In vivo tumors induced by C6­AS cells in nude mice exhibited a significant reduction in tumor growth compared with controls. This reduction could be mediated by the induction of MCH­I expression. The inhibition of glycogen synthesis by antisense glycogen synthase validates a putative target and a new approach for further study to advance the much­needed efficacy of intervention strategies for malignant gliomas.


Assuntos
Neoplasias Encefálicas/terapia , Terapia Genética , Glioma/terapia , Glicogênio Sintase/metabolismo , RNA Antissenso/uso terapêutico , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glioma/metabolismo , Glicogênio Sintase/química , Camundongos Nus , Invasividade Neoplásica , RNA Antissenso/farmacologia , Ratos , Receptor IGF Tipo 1/metabolismo
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