Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 99
Filtrar
1.
Mol Cell ; 72(4): 625-635.e4, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30454561

RESUMO

In response to genotoxic stress, cells activate a signaling cascade known as the DNA damage checkpoint (DDC) that leads to a temporary cell cycle arrest and activation of DNA repair mechanisms. Because persistent DDC activation compromises cell viability, this process must be tightly regulated. However, despite its importance, the mechanisms regulating DDC recovery are not completely understood. Here, we identify a DNA-damage-regulated histone modification in Saccharomyces cerevisiae, phosphorylation of H4 threonine 80 (H4T80ph), and show that it triggers checkpoint inactivation. H4T80ph is critical for cell survival to DNA damage, and its absence causes impaired DDC recovery and persistent cell cycle arrest. We show that, in response to genotoxic stress, p21-activated kinase Cla4 phosphorylates H4T80 to recruit Rtt107 to sites of DNA damage. Rtt107 displaces the checkpoint adaptor Rad9, thereby interrupting the checkpoint-signaling cascade. Collectively, our results indicate that H4T80ph regulates DDC recovery.


Assuntos
Dano ao DNA , Reparo do DNA , Histonas/genética , Histonas/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
2.
Genes Dev ; 32(17-18): 1242-1251, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30181361

RESUMO

During homologous recombination, cells must coordinate repair, DNA damage checkpoint signaling, and movement of chromosomal loci to facilitate homology search. In Saccharomyces cerevisiae, increased movement of damaged loci (local mobility) and undamaged loci (global mobility) precedes homolog pairing in mitotic cells. How cells modulate chromosome mobility in response to DNA damage remains unclear. Here, we demonstrate that global chromosome mobility is regulated by the Rad51 recombinase and its mediator, Rad52. Surprisingly, rad51Δ rad52Δ cells display checkpoint-dependent constitutively increased mobility, indicating that a regulatory circuit exists between recombination and checkpoint machineries to govern chromosomal mobility. We found that the requirement for Rad51 in this circuit is distinct from its role in recombination and that interaction with Rad52 is necessary to alleviate inhibition imposed by mediator recruitment to ssDNA. Thus, interplay between recombination factors and the checkpoint restricts increased mobility until recombination proteins are assembled at damaged sites.


Assuntos
Cromossomos Fúngicos/metabolismo , Dano ao DNA , Recombinação Homóloga , Rad51 Recombinase/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Mutação , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Genes Dev ; 32(23-24): 1499-1513, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30463903

RESUMO

In cells lacking telomerase, telomeres gradually shorten during each cell division to reach a critically short length, permanently activate the DNA damage checkpoint, and trigger replicative senescence. The increase in genome instability that occurs as a consequence may contribute to the early steps of tumorigenesis. However, because of the low frequency of mutations and the heterogeneity of telomere-induced senescence, the timing and mechanisms of genome instability increase remain elusive. Here, to capture early mutation events during replicative senescence, we used a combined microfluidic-based approach and live-cell imaging in yeast. We analyzed DNA damage checkpoint activation in consecutive cell divisions of individual cell lineages in telomerase-negative yeast cells and observed that prolonged checkpoint arrests occurred frequently in telomerase-negative lineages. Cells relied on the adaptation to the DNA damage pathway to bypass the prolonged checkpoint arrests, allowing further cell divisions despite the presence of unrepaired DNA damage. We demonstrate that the adaptation pathway is a major contributor to the genome instability induced during replicative senescence. Therefore, adaptation plays a critical role in shaping the dynamics of genome instability during replicative senescence.


Assuntos
Adaptação Fisiológica/genética , Pontos de Checagem do Ciclo Celular/genética , Dano ao DNA/genética , Instabilidade Genômica/genética , Saccharomyces cerevisiae/genética , Reparo do DNA , Genoma Fúngico/genética , Técnicas Analíticas Microfluídicas , Mutação , Imagem Óptica , Saccharomyces cerevisiae/enzimologia , Telomerase/genética
4.
J Biol Chem ; 300(3): 105751, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38354779

RESUMO

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Complexos Multiproteicos , Subunidades Proteicas , Humanos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Interações Hidrofóbicas e Hidrofílicas , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Domínios Proteicos
5.
Annu Rev Genet ; 50: 1-28, 2016 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-27732795

RESUMO

Double-strand breaks (DSBs) pose a severe challenge to genome integrity; consequently, cells have developed efficient mechanisms to repair DSBs through several pathways of homologous recombination and other nonhomologous end-joining processes. Much of our understanding of these pathways has come from the analysis of site-specific DSBs created by the HO endonuclease in the budding yeast Saccharomyces cerevisiae. I was fortunate to get in on the ground floor of analyzing the fate of synchronously induced DSBs through the study of what I coined "in vivo biochemistry." I have had the remarkable good fortune to profit from the development of new techniques that have permitted an ever more detailed dissection of these repair mechanisms, which are described here.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Reparo do DNA por Junção de Extremidades , Replicação do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Conversão Gênica , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Ácidos Nucleicos Heteroduplexes , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Genes Dev ; 30(10): 1211-24, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27222517

RESUMO

To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas Culina/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/genética , Fosforilação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38473708

RESUMO

DNA lesions trigger DNA damage checkpoint (DDC) signaling which arrests cell cycle progression and promotes DNA damage repair. In Saccharomyces cerevisiae, phosphorylation of histone H2A (γH2A, equivalent to γH2AX in mammals) is an early chromatin mark induced by DNA damage that is recognized by a group of DDC and DNA repair factors. We find that γH2A negatively regulates the G2/M checkpoint in response to the genotoxin camptothecin, which is a DNA topoisomerase I poison. γH2A also suppresses DDC signaling induced by the DNA alkylating agent methyl methanesulfonate. These results differ from prior findings, which demonstrate positive or no roles of γH2A in DDC in response to other DNA damaging agents such as phleomycin and ionizing radiation, which suggest that γH2A has DNA damage-specific effects on DDC signaling. We also find evidence supporting the notion that γH2A regulates DDC signaling by mediating the competitive recruitment of the DDC mediator Rad9 and the DNA repair factor Rtt107 to DNA lesions. We propose that γH2A/γH2AX serves to create a dynamic balance between DDC and DNA repair that is influenced by the nature of DNA damage.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dano ao DNA , Histonas/metabolismo , DNA/metabolismo
8.
Biochem Biophys Res Commun ; 685: 149157, 2023 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-37918324

RESUMO

During cell cycle progression in Saccharomyces cerevisiae, spindle pole bodies (SPBs) are duplicated during the G1/S-phase transition. SPBs are crucial for the organization of both the spindle and astral microtubules, and their orientation defines the direction of nuclear division. In this process, an old SPB, which serves as the template SPB during the duplication process, is oriented toward the bud side. The patterning microtubule plus-end tracking protein, Kar9, plays an important role in the orientation of SPBs by asymmetrically localizing to the old SPB. Here, methylglyoxal (MG), a metabolite derived from glycolysis, was found to perturb asymmetric Kar9 localization and influence proper positioning of the old SPB. MG activated the DNA damage checkpoint pathway, and MG-induced perturbation of asymmetric Kar9 localization was abolished by the deletion of MEC1, a sensor for the DNA damage checkpoint pathway. Methyl methanesulfonate, a DNA-alkylating agent, also perturbed asymmetric Kar9 localization. Our results suggest that activation of the DNA damage checkpoint pathway perturbs the asymmetric Kar9 localization required for proper positioning of SPBs.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Dano ao DNA , Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Corpos Polares do Fuso/metabolismo
9.
Mol Divers ; 27(6): 2789-2802, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36482226

RESUMO

DNA damage response (DDR) and autophagy are concerned with maintaining cellular homeostasis and dysregulation of these two pathways lead to pathologic conditions including tumorigenesis. Autophagy is activated as a protective mechanism during DDR which is indicative of their functional cooperativity but the molecular mechanism leading to the convergence of these two pathways during genotoxic stress remains elusive. In this study, through in silico analysis, we have shown an interaction between the Mediator of DNA damage checkpoint 1 (MDC1), an important DDR-associated protein, and Beclin-1, an autophagy inducer. MDC1 is an adaptor or scaffold protein known to regulate DDR, apoptosis, and cell cycle progression. While, Beclin-1 is involved in autophagosome nucleation and exhibits affinity for binding to Fork-head-associated domain (FHA) containing proteins. The FHA domain is commonly conserved in DDR-related proteins including MDC1. Through molecular docking, we have predicted the modeled complex between the MDC1 FHA domain and the Beclin-1 Coiled coil domain (CCD). The docking complex was modeled using ClusPro2.0, based on the crystal structure for the dimerized MDC1 FHA domain and Beclin-1 CCD. The complex stability and binding affinities were assessed using a Ramachandran plot, MD simulation, MM/GBSA, and PRODIGY webserver. Finally, the hot-spot residues at the interface were determined using computational alanine scanning by the DrugScorePPI webserver. Our analysis unveils significant interaction between MDC1 and Beclin-1, involving hydrogen bonds, non-bonded contacts, and salt bridges and indicates MDC1 possibly recruits Beclin-1 to the DSBs, as a consequence of which Beclin-1 is able to modulate DDR.


Assuntos
Proteínas de Ciclo Celular , Proteínas Nucleares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Beclina-1/metabolismo , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Simulação de Acoplamento Molecular , Autofagia
10.
Int J Mol Sci ; 24(6)2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36982663

RESUMO

We investigated the expression and biological function of retinoic acid inducible gene I (RIG-I) in esophageal squamous cell carcinoma (ESCC). Materials and methods: An immunohistochemical analysis was performed on 86 pairs of tumor tissue and adjacent normal tissue samples of patients with ESCC. We generated RIG-I-overexpressing ESCC cell lines KYSE70 and KYSE450, and RIG-I- knockdown cell lines KYSE150 and KYSE510. Cell viability, migration and invasion, radioresistance, DNA damage, and cell cycle were evaluated using CCK-8, wound-healing and transwell assay, colony formation, immunofluorescence, and flow cytometry and Western blotting, respectively. RNA sequencing was performed to determine the differential gene expression between controls and RIG-I knockdown. Tumor growth and radioresistance were assessed in nude mice using xenograft models. RIG-I expression was higher in ESCC tissues compared with that in matched non-tumor tissues. RIG-I overexpressing cells had a higher proliferation rate than RIG-I knockdown cells. Moreover, the knockdown of RIG-I slowed migration and invasion rates, whereas the overexpression of RIG-I accelerated migration and invasion rates. RIG-I overexpression induced radioresistance and G2/M phase arrest and reduced DNA damage after exposure to ionizing radiations compared with controls; however, it silenced the RIG-I enhanced radiosensitivity and DNA damage, and reduced the G2/M phase arrest. RNA sequencing revealed that the downstream genes DUSP6 and RIG-I had the same biological function; silencing DUSP6 can reduce the radioresistance caused by the overexpression of RIG-I. RIG-I knockdown depleted tumor growth in vivo, and radiation exposure effectively delayed the growth of xenograft tumors compared with the control group. RIG-I enhances the progression and radioresistance of ESCC; therefore, it may be a new potential target for ESCC-targeted therapy.


Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Animais , Humanos , Camundongos , Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Fosfatase 6 de Especificidade Dupla/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Receptores do Ácido Retinoico/metabolismo
11.
Int J Mol Sci ; 24(14)2023 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-37511442

RESUMO

The DNA damage response (DDR) is recognized as having an important role in cancer growth and treatment. ATR (ataxia telangiectasia mutated and Rad3-related) kinase, a major regulator of DDR, has shown significant therapeutic potential in cancer treatment. ATR inhibitors have shown anti-tumor effectiveness, not just as monotherapies but also in enhancing the effects of standard chemotherapy, radiation, and immunotherapy. The biological basis of ATR is examined in this review, as well as its functional significance in the development and therapy of cancer, and the justification for inhibiting this target as a therapeutic approach, including an assessment of the progress and status of previous decades' development of effective and selective ATR inhibitors. The current applications of these inhibitors in preclinical and clinical investigations as single medicines or in combination with chemotherapy, radiation, and immunotherapy are also fully reviewed. This review concludes with some insights into the many concerns highlighted or identified with ATR inhibitors in both the preclinical and clinical contexts, as well as potential remedies proposed.


Assuntos
Dano ao DNA , Neoplasias , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Carcinogênese/genética , Transformação Celular Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Desenvolvimento Embrionário
12.
Am J Physiol Cell Physiol ; 323(4): C1264-C1273, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36094439

RESUMO

In female mammals, the size of the initially established primordial follicle pool within the ovaries determines the reproductive life span. Interestingly, the establishment of the primordial follicle pool is accompanied by a remarkable programmed oocyte loss for unclear reasons. Here, we identify a new role of ASH1-like histone lysine methyltransferase (ASH1L) in controlling the apoptosis of oocytes during meiotic prophase I in mice. Our results showed that overexpression of Ash1l led to a dramatic loss of fetal oocytes via apoptosis, which subsequently resulted in a reduced capacity of the primordial follicle pool. Overexpression of Ash1l also led to a deficiency in DNA double-strand break repair associated with premature upregulation of p63 and phosphorylated checkpoint kinase 2 (p-CHK2), the major genome guardian of the female germline, following Ash1l overexpression in fetal ovaries. In summary, ASH1L is one of the indispensable epigenetic molecules that acts as a guardian of the genome. It protects oocyte genome integrity and removes oocytes with serious DNA damage by regulating the expression of p63 and p-CHK2 during meiotic prophase I in mice. Our study provides a perspective on the physiological regulatory role of DNA damage checkpoint signaling in fetal oocyte guardianship and female fertility.


Assuntos
Meiose , Oócitos , Animais , Apoptose/genética , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Mamíferos/metabolismo , Camundongos , Oócitos/metabolismo
13.
Mol Microbiol ; 116(2): 707-722, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34097787

RESUMO

During normal DNA replication, all cells encounter damage to their genetic material. As a result, organisms have developed response pathways that provide time for the cell to complete DNA repair before cell division occurs. In Bacillus subtilis, it is well established that the SOS-induced cell division inhibitor YneA blocks cell division after genotoxic stress; however, it remains unclear how YneA enforces the checkpoint. Here, we identify mutations that disrupt YneA activity and mutations that are refractory to the YneA-induced checkpoint. We find that YneA C-terminal truncation mutants and point mutants in or near the LysM peptidoglycan binding domain render YneA incapable of checkpoint enforcement. In addition, we develop a genetic method which isolated mutations in the ftsW gene that completely bypassed checkpoint enforcement while also finding that YneA interacts with late divisome components FtsL, Pbp2b, and Pbp1. Characterization of an FtsW variant resulted in considerably shorter cells during the DNA damage response indicative of hyperactive initiation of cell division and bypass of the YneA-enforced DNA damage checkpoint. With our results, we present a model where YneA inhibits septal cell wall synthesis by binding peptidoglycan and interfering with interaction between late arriving divisome components causing DNA damage checkpoint activation.


Assuntos
Bacillus subtilis/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Bacteriano/biossíntese , Peptidoglicano/biossíntese , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Divisão Celular/fisiologia , Dano ao DNA/genética , DNA Bacteriano/genética , Proteínas de Membrana/genética , Peptidoglicano/metabolismo
14.
EMBO J ; 37(9)2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29581097

RESUMO

Polymerase-blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single-stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re-priming downstream of lesions can give rise to daughter-strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9-dependent mechanism of damage signaling is distinct from the Mrc1-dependent, fork-associated response to replication stress induced by conditions such as nucleotide depletion or replisome-inherent problems, but reminiscent of replication-independent checkpoint activation by single-stranded DNA Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase-blocking lesions mainly emanates from Exo1-processed, postreplicative daughter-strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome- versus template-induced checkpoint signaling.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , DNA Fúngico/biossíntese , Fase S/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
J Biol Chem ; 295(4): 899-904, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31776186

RESUMO

DNA clamp, a highly conserved ring-shaped protein, binds dsDNA within its central pore. Also, DNA clamp interacts with various nuclear proteins on its front, thereby stimulating their enzymatic activities and biological functions. It has been assumed that the DNA clamp is a functionally single-faced ring from bacteria to humans. Here, we report the crystal structure of the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) checkpoint clamp bound to a peptide of RHINO, a recently identified cancer-related protein that interacts with 9-1-1 and promotes activation of the DNA damage checkpoint. This is the first structure of 9-1-1 bound to its partner. The structure reveals that RHINO is unexpectedly bound to the edge and around the back of the 9-1-1 ring through specific interactions with the RAD1 subunit of 9-1-1. Our finding indicates that 9-1-1 is a functionally double-faced DNA clamp.


Assuntos
Ciclo Celular , DNA/metabolismo , Peptídeos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Humanos , Modelos Moleculares , Peptídeos/química , Ligação Proteica
16.
Curr Genet ; 67(3): 439-445, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33620543

RESUMO

How cells respond to DNA damage is key to maintaining genome integrity or facilitating genetic change. In fungi, DNA damage responses have been extensively characterized in the model budding yeast Saccharomyces cerevisiae, which is generally not pathogenic. However, it is not clear how closely these responses resemble those in fungal pathogens, in which genetic change plays an important role in the evolutionary arms race between pathogen and host and the evolution of antifungal drug resistance. A close relative of S. cerevisiae, Candida glabrata, is an opportunistic pathogen that displays high variability in chromosome structure among clinical isolates and rapidly evolves antifungal drug resistance. The mechanisms facilitating such genomic flexibility and evolvability in this organism are unknown. Recently we characterized the DNA damage response of C. glabrata and identified several features that distinguish it from the well characterized DNA damage response of S. cerevisiae. First, we discovered that, in contrast to the established paradigm, C. glabrata effector kinase Rad53 is not hyperphosphorylated upon DNA damage. We also uncovered evidence of an attenuated DNA damage checkpoint response, wherein in the presence of DNA damage C. glabrata cells did not accumulate in S-phase and proceeded with cell division, leading to aberrant mitoses and cell death. Finally, we identified evidence of transcriptional rewiring of the DNA damage response of C. glabrata relative to S. cerevisiae, including an upregulation of genes involved in mating and meiosis-processes that have not been reported in C. glabrata. Together, these results open new possibilities and raise tantalizing questions of how this major fungal pathogen facilitates genetic change.


Assuntos
Candida glabrata/genética , Candidíase/genética , Dano ao DNA/genética , Variação Genética/genética , Candida glabrata/patogenicidade , Candidíase/microbiologia , Farmacorresistência Fúngica/genética , Genes Fúngicos Tipo Acasalamento/genética , Humanos , Meiose/genética
17.
Proc Natl Acad Sci U S A ; 115(51): E11961-E11969, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30510002

RESUMO

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5'-3' resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5' strands at DSB ends in a reaction stimulated by Sae2CtIP Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1-mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/metabolismo , DNA/metabolismo , Dano ao DNA , DNA Helicases , Reparo do DNA/fisiologia , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutagênicos , Proteínas Serina-Treonina Quinases/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
18.
J Biol Chem ; 294(8): 2690-2699, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30591583

RESUMO

The DNA damage response relies on protein modifications to elicit physiological changes required for coping with genotoxic conditions. Besides canonical DNA damage checkpoint-mediated phosphorylation, DNA damage-induced sumoylation has recently been shown to promote genotoxin survival. Cross-talk between these two pathways exists in both yeast and human cells. In particular, sumoylation is required for optimal checkpoint function, but the underlying mechanisms are not well-understood. To address this question, we examined the sumoylation of the first responder to DNA lesions, the ssDNA-binding protein complex replication protein A (RPA) in budding yeast (Saccharomyces cerevisiae). We delineated the sumoylation sites of the RPA large subunit, Rfa1 on the basis of previous and new mapping data. Findings using a sumoylation-defective Rfa1 mutant suggested that Rfa1 sumoylation acts in parallel with the 9-1-1 checkpoint complex to enhance the DNA damage checkpoint response. Mechanistically, sumoylated Rfa1 fostered an interaction with a checkpoint adaptor protein, Sgs1, and contributed to checkpoint kinase activation. Our results suggest that SUMO-based modulation of a DNA damage sensor positively influences the checkpoint response.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Quebras de DNA de Cadeia Simples , RecQ Helicases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sumoilação , Reparo do DNA , Fosforilação , Conformação Proteica , RecQ Helicases/genética , Proteína de Replicação A/química , Proteína de Replicação A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
19.
Curr Genet ; 66(1): 7-13, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31392389

RESUMO

The ribosomal RNA genes (rDNA) exist as tandem repeats in eukaryotes and are, therefore, highly unstable. Each rDNA unit includes a replication fork barrier site to avoid collisions between DNA replication forks and transcriptional machinery. However, because of this barrier, DNA double-strand breaks are induced at a relatively high frequency. If damage is repaired by the homologous recombination in rDNAs, it may result in frequent copy number changes and the production of extrachromosomal ribosomal DNA circles, both of which are closely linked to the regulation of lifespan. Here, we review recent progress in elucidating a multi-layered repair process of rDNA that occurs in the nucleolus, nucleoplasm and nuclear pores. Binding to nuclear pores appears to be the final strategy for repairing any remaining damage to the rDNA. Here, we propose the possible contribution of nuclear pores to the asymmetric distribution of damaged rDNA between mother and daughter cells as well as its possible impact on aging/rejuvenation.


Assuntos
Envelhecimento/genética , Duplicação Gênica , Genes de RNAr , Poro Nuclear/genética , Sequências de Repetição em Tandem , Animais , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Instabilidade Genômica , Humanos , Longevidade/genética , Poro Nuclear/metabolismo , Rejuvenescimento
20.
Curr Genet ; 66(1): 59-62, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31227863

RESUMO

DNA damage occurs abundantly during normal cellular proliferation. This necessitates that cellular DNA damage response and checkpoint pathways monitor the cellular DNA damage load and that DNA damage signaling is quantitative. Yet, how DNA lesions are counted and converted into a quantitative response remains poorly understood. We have recently obtained insights into this question investigating DNA damage signaling elicited by single-stranded DNA (ssDNA). Intriguingly, our findings suggest that local and global DNA damage signaling react differentially to increasing amounts of DNA damage. In this mini-review, we will discuss these findings and put them into perspective of current knowledge on the DNA damage response.


Assuntos
Dano ao DNA , Transdução de Sinais , Quebras de DNA de Cadeia Dupla , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Ligação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA