Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Más filtros













Base de datos
Intervalo de año de publicación
1.
PLoS Biol ; 22(3): e3002552, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38502677

RESUMEN

Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.


Asunto(s)
Replicación del ADN , ADN de Cadena Simple , Humanos , ADN de Cadena Simple/genética , Replicación del ADN/genética , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Unión Proteica , Ubiquitinación , Daño del ADN , Inestabilidad Genómica , ADN Helicasas/genética , ADN Helicasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
2.
J Proteome Res ; 22(8): 2765-2773, 2023 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-37463329

RESUMEN

Current protocols used to extract and purify histones are notoriously tedious, especially when using yeast cells. Here, we describe the use of a simple filter-aided sample preparation approach enabling histone extraction from yeast and mammalian cells using acidified ethanol, which not only improves extraction but also inactivates histone-modifying enzymes. We show that our improved method prevents N-terminal clipping of H3, an artifact frequently observed in yeast cells using standard histone extraction protocols. Our method is scalable and provides efficient recovery of histones when extracts are prepared from as few as two million yeast cells. We further demonstrate the application of this approach for the analysis of histone modifications in fungal clinical isolates available in a limited quantity. Compared with standard protocols, our method enables the study of histones and their modifications in a faster, simpler, and more robust manner.


Asunto(s)
Histonas , Saccharomyces cerevisiae , Animales , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Procesamiento Proteico-Postraduccional , Código de Histonas , Mamíferos/metabolismo
3.
J Biol Chem ; 299(7): 104900, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37301510

RESUMEN

Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.


Asunto(s)
Ciclina D1 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Reparación del ADN , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Humanos , Ciclina D1/genética , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/efectos de la radiación , Células HeLa , Proteínas Tirosina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fibroblastos/enzimología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Fase S , Fase G1 , Melanoma/genética , Melanoma/patología , Células Cultivadas , Rayos Ultravioleta/efectos adversos , Carcinogénesis/genética , Carcinogénesis/patología , Carcinogénesis/efectos de la radiación , Quinasas DyrK
4.
PLoS Biol ; 20(10): e3001543, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36215310

RESUMEN

Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RFs). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors that restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected "hits" acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identified SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI is recruited to UV-damaged chromatin and that its depletion promotes nascent DNA degradation at stalled RF. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro. Taken together, our data establish SCAI as a novel regulator of the UV-induced replicative stress response in human cells.


Asunto(s)
ADN de Cadena Simple , Proteína de Replicación A , Humanos , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , ADN de Cadena Simple/genética , Rayos Ultravioleta/efectos adversos , Replicación del ADN/genética , Cromatina , ADN , Mutágenos
5.
Mol Cell Biol ; 32(1): 154-72, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22025679

RESUMEN

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.


Asunto(s)
Daño del ADN , Replicación del ADN , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Antineoplásicos/farmacología , Camptotecina/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN , Replicación del ADN/efectos de los fármacos , ADN de Hongos/genética , ADN de Hongos/metabolismo , Histonas/genética , Hidroxiurea/farmacología , Lisina/genética , Metilmetanosulfonato/farmacología , Mutágenos/farmacología , Mutación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA