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1.
Mol Cell ; 65(5): 848-860.e11, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28257701

RESUMO

The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81's conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.


Assuntos
Reparo do DNA , Replicação do DNA , DNA/biossíntese , Endonucleases/metabolismo , Instabilidade Genômica , Ciclo Celular , DNA/química , DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/química , Endonucleases/genética , Ativação Enzimática , Humanos , Complexos Multienzimáticos , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Recombinases/genética , Recombinases/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo
2.
J Proteome Res ; 22(6): 1660-1681, 2023 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-37071664

RESUMO

The DNA repair scaffold SLX4 has pivotal roles in cellular processes that maintain genome stability, most notably homologous recombination. Germline mutations in SLX4 are associated with Fanconi anemia, a disease characterized by chromosome instability and cancer susceptibility. The role of mammalian SLX4 in homologous recombination depends critically on binding and activating structure-selective endonucleases, namely SLX1, MUS81-EME1, and XPF-ERCC1. Increasing evidence indicates that cells rely on distinct SLX4-dependent complexes to remove DNA lesions in specific regions of the genome. Despite our understanding of SLX4 as a scaffold for DNA repair proteins, a detailed repertoire of SLX4 interactors has never been reported. Here, we provide a comprehensive map of the human SLX4 interactome using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS). We identified 221 unique high-confidence interactors, of which the vast majority represent novel SLX4-binding proteins. Network analysis of these hits revealed pathways with known involvement of SLX4, such as DNA repair, and several emerging pathways of interest, including RNA metabolism and chromatin remodeling. In summary, the comprehensive SLX4 interactome we report here provides a deeper understanding of how SLX4 functions in DNA repair while revealing new cellular processes that may involve SLX4.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , DNA/genética , Recombinação Homóloga , Mamíferos/genética , Mamíferos/metabolismo , Recombinases/química , Recombinases/genética , Recombinases/metabolismo
3.
Microb Cell Fact ; 21(1): 280, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36587193

RESUMO

BACKGROUND: Over the 70 years since the introduction of plastic into everyday items, plastic waste has become an increasing problem. With over 360 million tonnes of plastics produced every year, solutions for plastic recycling and plastic waste reduction are sorely needed. Recently, multiple enzymes capable of degrading PET (polyethylene terephthalate) plastic have been identified and engineered. In particular, the enzymes PETase and MHETase from Ideonella sakaiensis depolymerize PET into the two building blocks used for its synthesis, ethylene glycol (EG) and terephthalic acid (TPA). Importantly, EG and TPA can be re-used for PET synthesis allowing complete and sustainable PET recycling. RESULTS: In this study we used Saccharomyces cerevisiae, a species utilized widely in bioindustrial fermentation processes, as a platform to develop a whole-cell catalyst expressing the MHETase enzyme, which converts monohydroxyethyl terephthalate (MHET) into TPA and EG. We assessed six expression architectures and identified those resulting in efficient MHETase expression on the yeast cell surface. We show that the MHETase whole-cell catalyst has activity comparable to recombinant MHETase purified from Escherichia coli. Finally, we demonstrate that surface displayed MHETase is active across a range of pHs, temperatures, and for at least 12 days at room temperature. CONCLUSIONS: We demonstrate the feasibility of using S. cerevisiae as a platform for the expression and surface display of PET degrading enzymes and predict that the whole-cell catalyst will be a viable alternative to protein purification-based approaches for plastic degradation.


Assuntos
Hidrolases , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Hidrolases/metabolismo , Etilenoglicol , Plásticos/metabolismo
4.
Mol Cell ; 52(2): 234-47, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24076221

RESUMO

Holliday junctions (HJs) are four-way DNA intermediates that form during homologous recombination, and their efficient resolution is essential for chromosome segregation. Here, we show that three structure-selective endonucleases, namely SLX1-SLX4, MUS81-EME1, and GEN1, define two pathways of HJ resolution in human cells. One pathway is mediated by GEN1, whereas SLX1-SLX4 and MUS81-EME1 provide a second and genetically distinct pathway (SLX-MUS). Cells depleted for SLX-MUS or GEN1 pathway proteins exhibit severe defects in chromosome segregation and reduced survival. In response to CDK-mediated phosphorylation, SLX1-SLX4 and MUS81-EME1 associate at the G2/M transition to form a stable SLX-MUS holoenzyme, which can be reconstituted in vitro. Biochemical studies show that SLX-MUS is a HJ resolvase that coordinates the active sites of two distinct endonucleases during HJ resolution. This cleavage reaction is more efficient and orchestrated than that mediated by SLX1-SLX4 alone, which exhibits a potent nickase activity that acts promiscuously upon DNA secondary structures.


Assuntos
DNA Cruciforme , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Recombinases/metabolismo , Sequência de Bases , Linhagem Celular Transformada , Reparo do DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Citometria de Fluxo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células HeLa , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Humanos , Immunoblotting , Modelos Genéticos , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Interferência de RNA , Recombinases/genética , Troca de Cromátide Irmã , Especificidade por Substrato
5.
Proc Natl Acad Sci U S A ; 114(3): 443-450, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-28049850

RESUMO

Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicase-TopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5'-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase, Escherichia coli RuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5'-A/TTTG/C-3'.


Assuntos
DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Resolvases de Junção Holliday/metabolismo , Sequência de Bases , Reparo do DNA , DNA Cruciforme/química , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Resolvases de Junção Holliday/química , Recombinação Homóloga , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
6.
STAR Protoc ; 4(2): 102152, 2023 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-36917604

RESUMO

Phosphorylation is a post-translational modification that can alter protein structure and regulate protein-protein interactions. Here, we present a procedure for in vitro phosphorylation of the MUS81-binding region of SLX4 (SLX4MBR) using cyclin-dependent kinase 1-cyclin B. We describe steps for the dialysis and phosphorylation of target proteins followed by purification using size-exclusion chromatography. Finally, we detail a system to monitor phosphorylation effectiveness and identify phosphorylated residues. We anticipate this protocol to be readily adapted for other protein targets or kinases. For complete details on the use and execution of this protocol, please refer to Payliss et al. (2022).1.

7.
Nucleic Acids Res ; 38(17): 5609-22, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20460453

RESUMO

The Nobel Prize in Physiology or Medicine was recently awarded to Elizabeth Blackburn, Carol Greider and Jack Szostak for their pioneering studies on chromosome termini (telomeres) and their discovery of telomerase, the enzyme that synthesizes telomeres. Telomerase is a unique cellular reverse transcriptase that contains an integral RNA subunit, the telomerase RNA and a catalytic protein subunit, the telomerase reverse transcriptase (TERT), as well as several species-specific accessory proteins. Telomerase is essential for genome stability and is associated with a broad spectrum of human diseases including various forms of cancer, bone marrow failure and pulmonary fibrosis. A better understanding of telomerase structure and function will shed important insights into how this enzyme contributes to human disease. To this end, a series of high-resolution structural studies have provided critical information on TERT architecture and may ultimately elucidate novel targets for therapeutic intervention. In this review, we discuss the current knowledge of TERT structure and function, revealed through the detailed analysis of TERT from model organisms. To emphasize the physiological importance of telomeres and telomerase, we also present a general discussion of the human diseases associated with telomerase dysfunction.


Assuntos
Telomerase/química , Telomerase/metabolismo , Humanos , Neoplasias/genética , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismo , Telomerase/genética , Telômero/metabolismo
8.
Cell Rep ; 41(4): 111537, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36288699

RESUMO

The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4MBR). Phosphorylated SLX4MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of SLX4MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. We also report the structure of phosphorylated SLX4MBR and identify the MUS81-binding interface. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.


Assuntos
Endonucleases , Recombinases , Endonucleases/metabolismo , Fosforilação , Recombinases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Reparo do DNA , Ciclina B/metabolismo , Endodesoxirribonucleases/metabolismo
9.
DNA Repair (Amst) ; 105: 103156, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139663

RESUMO

Nuclear reorganization, including the localization of proteins into discrete subnuclear foci, is a hallmark of the cellular response to DNA damage and replication stress. These foci are thought to represent transient environments or repair factories, in which the lesion is sequestered with molecules and co-factors that catalyze repair. For example, nuclear foci contain signaling proteins that recruit transducer proteins. One important class of transducers is the structure-selective endonucleases, such as SLX1-SLX4, MUS81-EME1, and XPF-ERCC1, which remove branched DNA structures that form during repair. The relocalization of structure-selective endonucleases into subnuclear foci provides a visual read-out for the presence of direct DNA damage, replication barriers, or DNA entanglements and can be monitored using fluorescence microscopy. By simultaneously probing for two or more fluorescent signals, fluorescence microscopy can also provide insights into the proximal association of proteins within a local environment. Here, we report an open-source and semi-automated method to detect and quantify subnuclear foci, as well as foci colocalization and the accompanying pixel-based colocalization metrics. We use this pipeline to show that pre-mitotic nuclei contain a basal threshold of foci marked by SLX1-SLX4, MUS81, or XPF. Some of these foci colocalize with FANCD2 and have a high degree of correlation and co-occurrence. We also show that pre-mitotic cells experiencing replication stress contain elevated levels of foci containing SLX1-SLX4 or XPF, but not MUS81. These results point towards a role for SLX1-SLX4 and XPF-ERCC1 in the early cellular response to replication stress. Nevertheless, most of the foci that form in response to replication stress contain either FANCD2 or one of the three endonucleases. Altogether, our work highlights the compositional heterogeneity of subnuclear foci that form in response to replication stress. We also describe a user-friendly pipeline that can be used to characterize these dynamic structures.


Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA , Testes de Mutagenicidade/métodos , Software , Linhagem Celular Tumoral , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Recombinases/metabolismo
10.
Front Genet ; 12: 784167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34804132

RESUMO

All organisms depend on the ability of cells to accurately duplicate and segregate DNA into progeny. However, DNA is frequently damaged by factors in the environment and from within cells. One of the most dangerous lesions is a DNA double-strand break. Unrepaired breaks are a major driving force for genome instability. Cells contain sophisticated DNA repair networks to counteract the harmful effects of genotoxic agents, thus safeguarding genome integrity. Homologous recombination is a high-fidelity, template-dependent DNA repair pathway essential for the accurate repair of DNA nicks, gaps and double-strand breaks. Accurate homologous recombination depends on the ability of cells to remove branched DNA structures that form during repair, which is achieved through the opposing actions of helicases and structure-selective endonucleases. This review focuses on a structure-selective endonuclease called SLX1-SLX4 and the macromolecular endonuclease complexes that assemble on the SLX4 scaffold. First, we discuss recent developments that illuminate the structure and biochemical properties of this somewhat atypical structure-selective endonuclease. We then summarize the multifaceted roles that are fulfilled by human SLX1-SLX4 and its associated endonucleases in homologous recombination and genome stability. Finally, we discuss recent work on SLX4-binding proteins that may represent integral components of these macromolecular nuclease complexes, emphasizing the structure and function of a protein called SLX4IP.

11.
Mol Cell Biol ; 27(8): 3226-40, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17296728

RESUMO

Telomerase is a ribonucleoprotein reverse transcriptase (RT) that processively synthesizes telomeric repeats onto the ends of linear chromosomes to maintain genomic stability. It has been proposed that the N terminus of the telomerase protein subunit, telomerase RT (TERT), contains an anchor site that forms stable interactions with DNA to prevent enzyme-DNA dissociation during translocation and to promote realignment events that accompany each round of telomere synthesis. However, it is not known whether human TERT (hTERT) can directly interact with DNA in the absence of the telomerase RNA subunit. Here we use a novel primer binding assay to establish that hTERT forms stable and specific contacts with telomeric DNA in the absence of the human telomerase RNA component (hTR). We show that hTERT-mediated primer binding can be functionally uncoupled from telomerase-mediated primer extension. Our results demonstrate that the first 350 amino acids of hTERT have a critical role in regulating the strength and specificity of protein-DNA interactions, providing additional evidence that the TERT N terminus contains an anchor site. Furthermore, we establish that the RT domain of hTERT mediates important protein-DNA interactions. Collectively, these data suggest that hTERT contains distinct anchor regions that cooperate to help regulate telomerase-mediated DNA recognition and elongation.


Assuntos
Telomerase/metabolismo , Aminoácidos , Sequência de Bases , Biotinilação , DNA/metabolismo , Primers do DNA , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Ligação Proteica , RNA/metabolismo , Telômero/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-26370409

RESUMO

DNA strand break repair by homologous recombination leads to the formation of intermediates in which sister chromatids are covalently linked. The efficient processing of these joint molecules, which often contain four-way structures known as Holliday junctions, is necessary for efficient chromosome segregation during mitotic division. Because persistent chromosome bridges pose a threat to genome stability, cells ensure the complete elimination of joint molecules through three independent pathways. These involve (1) BLM-Topoisomerase IIIα-RMI1-RMI2 (BTR complex), (2) SLX1-SLX4-MUS81-EME1 (SLX-MUS complex), and (3) GEN1. The BTR pathway promotes the dissolution of double Holliday junctions, which avoids the formation of crossover products, prevents sister chromatid exchanges, and limits the potential for loss of heterozygosity. In contrast to BTR, the other two pathways resolve Holliday junctions by nucleolytic cleavage to yield crossover and non-crossover products. To avoid competition with BTR, the resolution pathways are restrained until the late stages of the cell cycle. The temporal regulation of the dissolution/resolution pathways is therefore critical for crossover avoidance while also ensuring that all covalent links between chromosomes are resolved before chromosome segregation.


Assuntos
DNA Cruciforme/metabolismo , Reparo de DNA por Recombinação , Animais , Proteínas de Transporte/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Resolvases de Junção Holliday/metabolismo , Humanos , Perda de Heterozigosidade , Proteínas Nucleares/metabolismo , RecQ Helicases/metabolismo , Recombinases/metabolismo , Troca de Cromátide Irmã
13.
Cell Rep ; 10(9): 1467-1476, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25753413

RESUMO

The SLX1-SLX4 endonuclease required for homologous recombination and DNA repair in eukaryotic cells cleaves a variety of branched DNA structures. The nuclease subunit SLX1 is activated by association with a scaffolding protein SLX4. At the present time, little is known about the structure of SLX1-SLX4 or its mechanism of action. Here, we report the structural insights into SLX1-SLX4 by detailing the crystal structure of Candida glabrata (Cg) Slx1 alone and in combination with the C-terminal region of Slx4. The structure of Slx1 reveals a compact arrangement of the GIY-YIG nuclease and RING domains, which is reinforced by a long α helix. Slx1 forms a stable homodimer that blocks its active site. Slx1-Slx4 interaction is mutually exclusive with Slx1 homodimerization, suggesting a mechanism for Slx1 activation by Slx4.

14.
Cold Spring Harb Perspect Biol ; 6(9): a023192, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25183833

RESUMO

Four-way DNA intermediates, called Holliday junctions (HJs), can form during meiotic and mitotic recombination, and their removal is crucial for chromosome segregation. A group of ubiquitous and highly specialized structure-selective endonucleases catalyze the cleavage of HJs into two disconnected DNA duplexes in a reaction called HJ resolution. These enzymes, called HJ resolvases, have been identified in bacteria and their bacteriophages, archaea, and eukaryotes. In this review, we discuss fundamental aspects of the HJ structure and their interaction with junction-resolving enzymes. This is followed by a brief discussion of the eubacterial RuvABC enzymes, which provide the paradigm for HJ resolvases in other organisms. Finally, we review the biochemical and structural properties of some well-characterized resolvases from archaea, bacteriophage, and eukaryotes.


Assuntos
DNA Cruciforme/fisiologia , Resolvases de Junção Holliday/química , Archaea/enzimologia , Bactérias/enzimologia , Bacteriófagos/enzimologia , Núcleo Celular/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Resolvases de Junção Holliday/metabolismo , Humanos , Conformação Molecular , Multimerização Proteica , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
15.
Oncotarget ; 8(61): 102765-102766, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-29262521
16.
Aging Cell ; 11(3): 482-90, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22364217

RESUMO

Telomerase is a ribonucleoprotein reverse transcriptase (RT) that synthesizes specific DNA repeats, or telomeric DNA, at the ends of chromosomes. Telomerase is minimally composed of a protein subunit, TERT, and an RNA component, TR. Aberrant telomerase activity has been associated with most human cancers and several premature aging diseases, such as idiopathic pulmonary fibrosis (IPF), a chronic, progressive, and fatal lung disease characterized by alveolar epithelial cell damage and fibrosis. Our study focuses on three hTERT mutations that were identified in a subset of patients with IPF, in which these patients also exhibited shorter telomeres compared with age-matched controls. We characterized how three IPF-associated hTERT mutations, V144M, R865C, and R865H, affected telomerase function both in vitro and in human cells. We demonstrated that the R865 residue is crucial for repeat addition processivity and thus telomere synthesis in telomerase-positive 293 cells and telomerase-negative BJ cells, consistent with its location in the hTERT nucleotide-binding motif. In contrast, while the V144M mutant did not exhibit any biochemical defects, this mutant was unable to elongate telomeres in human cells. As a result, our studies have identified hTERT V144 and R865 as two critical residues required for proper telomerase function in cells. Together, this may explain how inherited hTERT mutations can lead to shortened telomeres in patients with IPF and, thus, provide further insight into the role of naturally occurring telomerase mutations in the pathophysiology of certain age-related disease states.


Assuntos
Fibrose Pulmonar Idiopática/enzimologia , Fibrose Pulmonar Idiopática/genética , Mutação , Telomerase/genética , Telomerase/metabolismo , Idoso de 80 Anos ou mais , Senilidade Prematura/enzimologia , Senilidade Prematura/genética , Senilidade Prematura/metabolismo , Sequência de Aminoácidos , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Fibroblastos/citologia , Humanos , Fibrose Pulmonar Idiopática/patologia , Rim/citologia , Dados de Sequência Molecular
17.
PLoS One ; 4(9): e7176, 2009 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-19777057

RESUMO

BACKGROUND: Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE: We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention.


Assuntos
Telomerase/metabolismo , Catálise , Linhagem Celular , Linhagem Celular Transformada , DNA/química , Glutamina/química , Humanos , Técnicas In Vitro , Mutação , Oligonucleotídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , RNA/química , Telomerase/química , Telomerase/genética , Telomerase/fisiologia
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