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1.
Mol Cell ; 84(14): 2598-2600, 2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-39059369

RESUMO

Recently in Cell, Cai et al.1 reported how phosphorylation of human shelterin protein POT1 allows it to recruit the telomeric C-rich strand replication machinery, providing mechanistic insights into an understudied area of telomere biology with implications for telomere biology disorders.


Assuntos
Replicação do DNA , Complexo Shelterina , Proteínas de Ligação a Telômeros , Telômero , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ligação a Telômeros/genética , Complexo Shelterina/metabolismo , Humanos , Fosforilação , Telômero/metabolismo , Telômero/genética
2.
Proc Natl Acad Sci U S A ; 121(16): e2316651121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38588418

RESUMO

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.


Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Ligação a Telômeros , Animais , Proteínas de Ligação a Telômeros/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dimerização , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/química , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Ligação Proteica , Telômero/genética , Telômero/metabolismo , Complexo Shelterina , DNA/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas , Mamíferos/genética
3.
Trends Biochem Sci ; 47(6): 506-517, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35440402

RESUMO

Telomeres are chromosome-capping structures that protect ends of the linear genome from DNA damage sensors. However, these structures present obstacles during DNA replication. Incomplete telomere replication accelerates telomere shortening and limits replicative lifespan. Therefore, continued proliferation under conditions of replication stress requires a means of telomere repair, particularly in the absence of telomerase. It was recently revealed that replication stress triggers break-induced replication (BIR) and mitotic DNA synthesis (MiDAS) at mammalian telomeres; however, these mechanisms are error prone and primarily utilized in tumorigenic contexts. In this review article, we discuss the consequences of replication stress at telomeres and how use of available repair pathways contributes to genomic instability. Current research suggests that fragile telomeres are ultimately tumor-suppressive and thus may be better left unrepaired.


Assuntos
Telomerase , Telômero , Animais , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Mamíferos , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Homeostase do Telômero
4.
Nucleic Acids Res ; 52(11): 6347-6359, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38661211

RESUMO

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , Magnésio , Mitomicina , Mitomicina/farmacologia , Mitomicina/química , Magnésio/química , Magnésio/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Modelos Moleculares , Domínio Catalítico , Reparo do DNA , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Cristalografia por Raios X , DNA/metabolismo , DNA/química , Exonucleases/metabolismo , Exonucleases/química
5.
Nat Rev Mol Cell Biol ; 14(2): 69-82, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23299958

RESUMO

Telomeres, the ends of linear eukaryotic chromosomes, are characterized by the presence of multiple repeats of a short DNA sequence. This telomeric DNA is protected from illicit repair by telomere-associated proteins, which in mammals form the shelterin complex. Replicative polymerases are unable to synthesize DNA at the extreme ends of chromosomes, but in unicellular eukaryotes such as yeast and in mammalian germ cells and stem cells, telomere length is maintained by a ribonucleoprotein enzyme known as telomerase. Recent work has provided insights into the mechanisms of telomerase recruitment to telomeres, highlighting the contribution of telomere-associated proteins, including TPP1 in humans, Ccq1 in Schizosaccharomyces pombe and Cdc13 and Ku70-Ku80 in Saccharomyces cerevisiae.


Assuntos
Cromossomos/química , Telomerase/metabolismo , Telômero/metabolismo , Animais , Cromossomos/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Complexo Shelterina , Telômero/química , Telômero/genética , Proteínas de Ligação a Telômeros
6.
PLoS Genet ; 18(5): e1010196, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35576203

RESUMO

CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over-initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 Å resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation.


Assuntos
Proteínas de Ligação a DNA , Ribose , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Ciclo Celular/genética , Colina/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Ribose/metabolismo
7.
Nucleic Acids Res ; 50(16): 9413-9425, 2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-36018809

RESUMO

Mutations in DKC1 (encoding dyskerin) cause telomere diseases including dyskeratosis congenita (DC) by decreasing steady-state levels of TERC, the non-coding RNA component of telomerase. How DKC1 mutations variably impact numerous other snoRNAs remains unclear, which is a barrier to understanding disease mechanisms in DC beyond impaired telomere maintenance. Here, using DC patient iPSCs, we show that mutations in the dyskerin N-terminal extension domain (NTE) dysregulate scaRNA13. In iPSCs carrying the del37L NTE mutation or engineered to carry NTE mutations via CRISPR/Cas9, but not in those with C-terminal mutations, we found scaRNA13 transcripts with aberrant 3' extensions, as seen when the exoribonuclease PARN is mutated in DC. Biogenesis of scaRNA13 was rescued by repair of the del37L DKC1 mutation by genome-editing, or genetic or pharmacological inactivation of the polymerase PAPD5, which counteracts PARN. Inspection of the human telomerase cryo-EM structure revealed that in addition to mediating intermolecular dyskerin interactions, the NTE interacts with terminal residues of the associated snoRNA, indicating a role for this domain in 3' end definition. Our results provide mechanistic insights into the interplay of dyskerin and the PARN/PAPD5 axis in the biogenesis and accumulation of snoRNAs beyond TERC, broadening our understanding of ncRNA dysregulation in human diseases.


Assuntos
Disceratose Congênita , Telomerase , Humanos , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Disceratose Congênita/genética , Mutação , Proteínas de Ligação a RNA/genética
8.
Blood ; 138(10): 898-911, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34019641

RESUMO

Germline pathogenic TERT variants are associated with short telomeres and an increased risk of developing myelodysplastic syndrome (MDS) among patients with a telomere biology disorder. We identified TERT rare variants in 41 of 1514 MDS patients (2.7%) without a clinical diagnosis of a telomere biology disorder who underwent allogeneic transplantation. Patients with a TERT rare variant had shorter telomere length (P < .001) and younger age at MDS diagnosis (52 vs 59 years, P = .03) than patients without a TERT rare variant. In multivariable models, TERT rare variants were associated with inferior overall survival (P = .034) driven by an increased incidence of nonrelapse mortality (NRM; P = .015). Death from a noninfectious pulmonary cause was more frequent among patients with a TERT rare variant. Most variants were missense substitutions and classified as variants of unknown significance. Therefore, we cloned all rare missense variants and quantified their impact on telomere elongation in a cell-based assay. We found that 90% of TERT rare variants had severe or intermediate impairment in their capacity to elongate telomeres. Using a homology model of human TERT bound to the shelterin protein TPP1, we inferred that TERT rare variants disrupt domain-specific functions, including catalysis, protein-RNA interactions, and recruitment to telomeres. Our results indicate that the contribution of TERT rare variants to MDS pathogenesis and NRM risk is underrecognized. Routine screening for TERT rare variants in MDS patients regardless of age or clinical suspicion may identify clinically inapparent telomere biology disorders and improve transplant outcomes through risk-adapted approaches.


Assuntos
Variação Genética , Síndromes Mielodisplásicas , Telomerase/genética , Adulto , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Síndromes Mielodisplásicas/enzimologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Taxa de Sobrevida
9.
J Biol Chem ; 296: 100064, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33482595

RESUMO

Genetic mutations that affect telomerase function or telomere maintenance result in a variety of diseases collectively called telomeropathies. This wide spectrum of disorders, which include dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia, is characterized by severely short telomeres, often resulting in hematopoietic stem cell failure in the most severe cases. Recent work has focused on understanding the molecular basis of these diseases. Mutations in the catalytic TERT and TR subunits of telomerase compromise activity, while others, such as those found in the telomeric protein TPP1, reduce the recruitment of telomerase to the telomere. Mutant telomerase-associated proteins TCAB1 and dyskerin and the telomerase RNA maturation component poly(A)-specific ribonuclease affect the maturation and stability of telomerase. In contrast, disease-associated mutations in either CTC1 or RTEL1 are more broadly associated with telomere replication defects. Yet even with the recent surge in studies decoding the mechanisms underlying these diseases, a significant proportion of dyskeratosis congenita mutations remain uncharacterized or poorly understood. Here we review the current understanding of the molecular basis of telomeropathies and highlight experimental data that illustrate how genetic mutations drive telomere shortening and dysfunction in these patients. This review connects insights from both clinical and molecular studies to create a comprehensive view of the underlying mechanisms that drive these diseases. Through this, we emphasize recent advances in therapeutics and pinpoint disease-associated variants that remain poorly defined in their mechanism of action. Finally, we suggest future avenues of research that will deepen our understanding of telomere biology and telomere-related disease.


Assuntos
Telômero , Anemia Aplástica/genética , Disceratose Congênita/genética , Humanos , Mutação , Complexo Shelterina , Telomerase/genética , Telomerase/metabolismo , Encurtamento do Telômero , Proteínas de Ligação a Telômeros
10.
Proc Natl Acad Sci U S A ; 116(52): 26505-26515, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31822618

RESUMO

Telomerase catalyzes telomeric DNA synthesis at chromosome ends to allow for continued cell division. The telomeric protein TPP1 is essential for enhancing the processivity of telomerase and recruiting the enzyme to telomeres. The telomerase interaction surface on human TPP1 has been mapped to 2 regions of the N-terminal oligosaccharide/oligonucleotide-binding (OB) domain, namely the TPP1 glutamate (E) and leucine (L)-rich (TEL) patch and the N terminus of TPP1-oligosaccharide/oligonucleotide-binding (NOB) region. To map the telomerase side of the interface, we exploited the predicted structural similarities for human and Tetrahymena thermophila telomerase as well as the species specificity of human and mouse telomerase for their cognate TPP1 partners. We show that swapping in the telomerase essential N-terminal (TEN) and insertions in fingers domain (IFD)-TRAP regions of the human telomerase catalytic protein subunit TERT into the mouse TERT backbone is sufficient to bias the species specificity toward human TPP1. Employing a structural homology-based mutagenesis screen focused on surface residues of the TEN and IFD regions, we identified TERT residues that are critical for contacting TPP1 but dispensable for other aspects of telomerase structure or function. We present a functionally validated structural model for how human telomerase engages TPP1 at telomeres, setting the stage for a high-resolution structure of this interface.

11.
Genes Dev ; 28(19): 2090-102, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25233904

RESUMO

Germline mutations in telomere biology genes cause dyskeratosis congenita (DC), an inherited bone marrow failure and cancer predisposition syndrome. DC is a clinically heterogeneous disorder diagnosed by the triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia; Hoyeraal-Hreidarsson syndrome (HH), a clinically severe variant of DC, also includes cerebellar hypoplasia, immunodeficiency, and intrauterine growth retardation. Approximately 70% of DC cases are associated with a germline mutation in one of nine genes, the products of which are all involved in telomere biology. Using exome sequencing, we identified mutations in Adrenocortical Dysplasia Homolog (ACD) (encoding TPP1), a component of the telomeric shelterin complex, in one family affected by HH. The proband inherited a deletion from his father and a missense mutation from his mother, resulting in extremely short telomeres and a severe clinical phenotype. Characterization of the mutations revealed that the single-amino-acid deletion affecting the TEL patch surface of the TPP1 protein significantly compromises both telomerase recruitment and processivity, while the missense mutation in the TIN2-binding region of TPP1 is not as clearly deleterious to TPP1 function. Our results emphasize the critical roles of the TEL patch in proper stem cell function and demonstrate that TPP1 is the second shelterin component (in addition to TIN2) to be implicated in DC.


Assuntos
Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Mutação em Linhagem Germinativa/genética , Deficiência Intelectual/genética , Microcefalia/genética , Serina Proteases/genética , Adulto , Criança , Pré-Escolar , Disceratose Congênita/patologia , Feminino , Retardo do Crescimento Fetal/patologia , Células HeLa , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Microcefalia/patologia , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Linhagem , Estrutura Terciária de Proteína , Deleção de Sequência/genética , Serina Proteases/química , Complexo Shelterina , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo
12.
Cell Mol Life Sci ; 77(1): 61-79, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31728577

RESUMO

Telomeres are protein-DNA complexes that protect chromosome ends from illicit ligation and resection. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA to counter telomere shortening. Human telomeres are composed of complexes between telomeric DNA and a six-protein complex known as shelterin. The shelterin proteins TRF1 and TRF2 provide the binding affinity and specificity for double-stranded telomeric DNA, while the POT1-TPP1 shelterin subcomplex coats the single-stranded telomeric G-rich overhang that is characteristic of all our chromosome ends. By capping chromosome ends, shelterin protects telomeric DNA from unwanted degradation and end-to-end fusion events. Structures of the human shelterin proteins reveal a network of constitutive and context-specific interactions. The shelterin protein-DNA structures reveal the basis for both the high affinity and DNA sequence specificity of these interactions, and explain how shelterin efficiently protects chromosome ends from genome instability. Several protein-protein interactions, many provided by the shelterin component TIN2, are critical for upholding the end-protection function of shelterin. A survey of these protein-protein interfaces within shelterin reveals a series of "domain-peptide" interactions that allow for efficient binding and adaptability towards new functions. While the modular nature of shelterin has facilitated its part-by-part structural characterization, the interdependence of subunits within telomerase has made its structural solution more challenging. However, the exploitation of several homologs in combination with recent advancements in cryo-EM capabilities has led to an exponential increase in our knowledge of the structural biology underlying telomerase function. Telomerase homologs from a wide range of eukaryotes show a typical retroviral reverse transcriptase-like protein core reinforced with elements that deliver telomerase-specific functions including recruitment to telomeres and high telomere-repeat addition processivity. In addition to providing the template for reverse transcription, the RNA component of telomerase provides a scaffold for the catalytic and accessory protein subunits, defines the limits of the telomeric repeat sequence, and plays a critical role in RNP assembly, stability, and trafficking. While a high-resolution definition of the human telomerase structure is only beginning to emerge, the quick pace of technical progress forecasts imminent breakthroughs in this area. Here, we review the structural biology surrounding telomeres and telomerase to provide a molecular description of mammalian chromosome end protection and end replication.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Aminopeptidases/química , Aminopeptidases/metabolismo , Animais , Cromossomos/química , Cromossomos/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Serina Proteases/química , Serina Proteases/metabolismo , Complexo Shelterina , Telomerase/química , Telômero/química , Proteínas de Ligação a Telômeros/química
13.
Proc Natl Acad Sci U S A ; 113(46): 13021-13026, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27807141

RESUMO

Telomerase replicates chromosome ends to facilitate continued cell division. Mutations that compromise telomerase function result in stem cell failure diseases, such as dyskeratosis congenita (DC). One such mutation (K170Δ), residing in the telomerase-recruitment factor TPP1, provides an excellent opportunity to structurally, biochemically, and genetically dissect the mechanism of such diseases. We show through site-directed mutagenesis and X-ray crystallography that this TPP1 disease mutation deforms the conformation of two critical amino acids of the TEL [TPP1's glutamate (E) and leucine-rich (L)] patch, the surface of TPP1 that binds telomerase. Using CRISPR-Cas9 technology, we demonstrate that introduction of this mutation in a heterozygous manner is sufficient to shorten telomeres in human cells. Our findings rule out dominant-negative effects of the mutation. Instead, these findings implicate reduced TEL patch dosage in causing telomere shortening. Our studies provide mechanistic insight into telomerase-deficiency diseases and encourage the development of gene therapies to counter such diseases.


Assuntos
Disceratose Congênita/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética , Linhagem Celular Tumoral , Cristalografia por Raios X , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Complexo Shelterina , Telomerase/metabolismo , Telômero/metabolismo
14.
Nature ; 492(7428): 285-9, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23103865

RESUMO

Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase. Shelterin includes the heterodimeric POT1-TPP1 protein, which binds the telomeric single-stranded DNA tail. TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-binding event). Determining the mechanisms of these activities has been difficult, especially because genetic perturbations also tend to affect the essential chromosome end-protection function of TPP1 (refs 15-17). Here we identify separation-of-function mutants of human TPP1 that retain full telomere-capping function in vitro and in vivo, yet are defective in binding human telomerase. The seven separation-of-function mutations map to a patch of amino acids on the surface of TPP1, the TEL patch, that both recruits telomerase to telomeres and promotes high-processivity DNA synthesis, indicating that these two activities are manifestations of the same molecular interaction. Given that the interaction between telomerase and TPP1 is required for telomerase function in vivo, the TEL patch of TPP1 provides a new target for anticancer drug development.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Complexo Shelterina , Telômero/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética
15.
Genes Dev ; 24(6): 613-22, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20231318

RESUMO

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3' ssDNA extension of human telomeres. POT1-TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1-TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1-TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1-TPP1, in contrast to other single amino acid mutations. Chimeric human-fish telomerases that contained the human TEN domain were active but not stimulated by POT1-TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A-mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1-POT1-telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/genética , Humanos , Camundongos , Modelos Moleculares , Mutação/genética , Oryzias , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética
16.
Anal Biochem ; 530: 40-49, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28477963

RESUMO

CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci.


Assuntos
Proteínas Argonautas/antagonistas & inibidores , Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes/métodos , Marcação de Genes , Lentivirus/genética , RNA Guia de Cinetoplastídeos/genética , Proteínas Argonautas/genética , Células HeLa , Humanos , Fenótipo
17.
Mol Cell ; 31(2): 278-86, 2008 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-18657509

RESUMO

RNA healing and sealing enzymes drive informational and stress response pathways entailing repair of programmed 2',3' cyclic PO(4)/5'-OH breaks. Fungal, plant, and phage tRNA ligases use different strategies to discriminate the purposefully broken ends of the anticodon loop. Whereas phage ligase recognizes the tRNA fold, yeast and plant ligases do not and are instead hardwired to seal only the tRNA 3'-OH, 2'-PO(4) ends formed by healing of a cyclic phosphate. tRNA anticodon damage inflicted by secreted ribotoxins such as fungal gamma-toxin underlies a rudimentary innate immune system. Yeast cells are susceptible to gamma-toxin because the sealing domain of yeast tRNA ligase is unable to rectify a break at the modified wobble base of tRNA(Glu(UUC)). Plant andphage tRNA repair enzymes protect yeast from gamma-toxin because they are able to reverse the damage. Our studies underscore how a ribotoxin exploits an Achilles' heel in the target cell's tRNA repair system.


Assuntos
Antídotos/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , RNA de Transferência/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Bacteriófagos/enzimologia , Sequência de Bases , Morte Celular/efeitos dos fármacos , Células Eucarióticas/efeitos dos fármacos , Fatores Matadores de Levedura , Dados de Sequência Molecular , Micotoxinas/toxicidade , Conformação de Ácido Nucleico/efeitos dos fármacos , RNA Ligase (ATP)/metabolismo , RNA Fúngico/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência de Ácido Glutâmico/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Toxinas Biológicas/toxicidade
18.
J Biol Chem ; 288(46): 33171-80, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24097987

RESUMO

Continued proliferation of human cells requires maintenance of telomere length, usually accomplished by telomerase. Telomerase is recruited to chromosome ends by interaction with a patch of amino acids (the TEL patch, for TPP1 glutamate (E) and leucine (L)-rich patch) on the surface of telomere protein TPP1. In previous studies, interruption of this interaction by mutation prevented telomere extension in HeLa cells, but the cell culture continued to grow. We now show that the telomerase inhibitor BIBR1532 acts together with TEL patch mutations to inhibit the growth of HeLa cell lines and that apoptosis is a prominent mechanism of death of these cells. Survivor cells take over the population beginning around 40 days in culture. These cells no longer express the TEL patch mutant TPP1, apparently because of silencing of the expression cassette, a survival mechanism that would not be available to cancer cells. These results provide hope that inhibiting the binding of telomerase to the TEL patch of TPP1, perhaps together with a modest inhibition of the telomerase enzyme, could comprise an effective anticancer therapy for the ∼90% of human tumors that are telomerase-positive.


Assuntos
Aminobenzoatos/farmacologia , Apoptose/efeitos dos fármacos , Naftalenos/farmacologia , Neoplasias/enzimologia , Telomerase/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Serina Proteases/genética , Serina Proteases/metabolismo , Complexo Shelterina , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros
19.
Nucleic Acids Res ; 40(1): 235-44, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21911358

RESUMO

Eukaryotic chromosome ends are protected from illicit DNA joining by protein-DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA , Dimerização , Oligonucleotídeos/química , Estrutura Terciária de Proteína , RNA/química , Sequências Repetitivas de Ácido Nucleico , Proteínas de Schizosaccharomyces pombe/química , Complexo Shelterina , Telômero/química , Proteínas de Ligação a Telômeros/química , Timidina/química , Uridina/química
20.
bioRxiv ; 2024 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-38405983

RESUMO

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

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