RESUMO
Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamentally valuable structural information regarding telomere biology.
Assuntos
Proteínas de Ligação a DNA/química , Simulação de Acoplamento Molecular , Proteínas de Saccharomyces cerevisiae/química , Telomerase/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Premature telomere shortening or telomere instability is associated with a group of rare and heterogeneous diseases collectively known as telomere biology disorders (TBDs). Here we identified two unrelated individuals with clinical manifestations of TBDs and short telomeres associated with the identical monoallelic variant c.767A>G; Y256C in RPA2 Although the replication protein A2 (RPA2) mutant did not affect ssDNA binding and G-quadruplex-unfolding properties of RPA, the mutation reduced the affinity of RPA2 with the ubiquitin ligase RFWD3 and reduced RPA ubiquitination. Using engineered knock-in cell lines, we found an accumulation of RPA at telomeres that did not trigger ATR activation but caused short and dysfunctional telomeres. Finally, both patients acquired, in a subset of blood cells, somatic genetic rescue events in either POT1 genes or TERT promoters known to counteract the accelerated telomere shortening. Collectively, our study indicates that variants in RPA2 represent a novel genetic cause of TBDs. Our results further support the fundamental role of the RPA complex in regulating telomere length and stability in humans.
RESUMO
The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.
Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Replicação do DNA/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , Nucleossomos/genética , RecQ Helicases/genética , Saccharomyces cerevisiae/genética , CoesinasRESUMO
As in human cells, yeast telomeres can be maintained in cells lacking telomerase activity by recombination-based mechanisms known as ALT (Alternative Lengthening of Telomeres). A hallmark of ALT human cancer cells are extrachromosomal telomeric DNA elements called C-circles, whose origin and function have remained unclear. Here, we show that extrachromosomal telomeric C-circles in yeast can be detected shortly after senescence crisis and concomitantly with the production of survivors arising from "type II" recombination events. We uncover that C-circles bind to the nuclear pore complex (NPC) and to the SAGA-TREX2 complex, similar to other non-centromeric episomal DNA. Disrupting the integrity of the SAGA/TREX2 complex affects both C-circle binding to NPCs and type II telomere recombination, suggesting that NPC tethering of C-circles facilitates formation and/or propagation of the long telomere repeats characteristic of type II survivors. Furthermore, we find that disruption of the nuclear diffusion barrier impairs type II recombination. These results support a model in which concentration of C-circles at NPCs benefits type II telomere recombination, highlighting the importance of spatial coordination in ALT-type mechanisms of telomere maintenance.
Assuntos
Poro Nuclear , Saccharomyces cerevisiae , Citoplasma , Humanos , Poro Nuclear/genética , Saccharomyces cerevisiae/genética , Telômero/genéticaRESUMO
Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.
Assuntos
Enfisema , Rarefação Microvascular , Enfisema Pulmonar , Telomerase , Camundongos , Animais , Encurtamento do Telômero , Telomerase/genéticaRESUMO
R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA-DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA-DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo. Importantly, an excess of Yra1 increases R-loop-mediated genome instability caused by transcription-replication collisions regardless of whether they are codirectional or head-on. It also induces telomere shortening in telomerase-negative cells and accelerates senescence, consistent with a defect in telomere replication. Our results indicate that RNA-DNA hybrids form transiently in cells regardless of replication and, after stabilization by excess Yra1, compromise genome integrity, in agreement with a two-step model of R-loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.
Assuntos
Instabilidade Cromossômica/genética , Replicação do DNA , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/genética , Transcrição Gênica , Cromatina/metabolismo , Hibridização de Ácido Nucleico , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismoRESUMO
Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage.
Assuntos
DNA Fúngico/genética , Instabilidade Genômica , Íntrons , Ácidos Nucleicos Heteroduplexes/genética , RNA Fúngico/genética , Transcrição Gênica , Candida glabrata/genética , Candida glabrata/metabolismo , Linhagem Celular , Biologia Computacional , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Dano ao DNA , DNA Fúngico/química , DNA Fúngico/metabolismo , Bases de Dados Genéticas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genótipo , Humanos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/metabolismo , Fenótipo , Splicing de RNA , RNA Fúngico/química , RNA Fúngico/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
Inherited bone marrow failure syndromes (IBMFSs) are a group of disorders typified by impaired production of 1 or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant, Høyeraal-Hreidarsson (HH) syndrome, are rare IBMFSs characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. We identified biallelic variants in the gene encoding the 5'-to-3' DNA exonuclease Apollo/SNM1B in 3 unrelated patients presenting with a DC/HH phenotype consisting of early-onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly, and/or intrauterine growth retardation. All 3 patients carry a homozygous or compound heterozygous (in combination with a null allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients' cells showed signs of telomere fragility that were not associated with global reduction of telomere length. Unlike patients' cells, human Apollo KO HT1080 cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollo variants as a genetic cause of a hitherto unrecognized severe IBMFS that combines clinical hallmarks of DC/HH with normal telomere length.
Assuntos
Disceratose Congênita , Deficiência Intelectual , Microcefalia , Disceratose Congênita/genética , Disceratose Congênita/metabolismo , Retardo do Crescimento Fetal , Humanos , Deficiência Intelectual/genética , Microcefalia/genética , Microcefalia/metabolismo , Mutação , Telômero/genética , Telômero/metabolismoRESUMO
Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.
Assuntos
Transtornos da Insuficiência da Medula Óssea/patologia , Mutação com Ganho de Função , Heterozigoto , Síndromes Mielodisplásicas/patologia , Proteína de Replicação A/genética , Encurtamento do Telômero , Telômero/genética , Adolescente , Adulto , Transtornos da Insuficiência da Medula Óssea/etiologia , Transtornos da Insuficiência da Medula Óssea/metabolismo , Diferenciação Celular , Criança , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/metabolismo , Adulto JovemRESUMO
Telomere elongation is coupled with genome replication, raising the question of the repair of short telomeres in post-mitotic cells. We investigated the fate of a telomere-repeat capped end that mimics a single short telomere in quiescent fission yeast cells. We show that telomerase is able to elongate this single short telomere during quiescence despite the binding of Ku to the proto-telomere. While Taz1 and Rap1 repress telomerase in vegetative cells, both shelterin proteins are required for efficient telomere extension in quiescent cells, underscoring a distinct mode of telomerase control. We further show that Rad3ATR and Tel1ATM are redundantly required for telomere elongation in quiescence through the phosphorylation of Ccq1 and that Rif1 and its associated-PP1 phosphatases negatively regulate telomerase activity by opposing Ccq1 phosphorylation. The distinct mode of telomerase regulation in quiescent fission yeast cells may be relevant to that in human stem and progenitor cells.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Complexo Shelterina , Telomerase , Proteínas de Ligação a Telômeros , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Telomere anchoring to nuclear envelope (NE) is a key feature of nuclear genome architecture. Peripheral localization of telomeres is important for chromatin silencing, telomere replication and for the control of inappropriate recombination. Here, we report that fission yeast quiescent cells harbor predominantly a single telomeric cluster anchored to the NE. Telomere cluster association to the NE relies on Rap1-Bqt4 interaction, which is impacted by the length of telomeric sequences. In quiescent cells, reducing telomere length or deleting bqt4, both result in an increase in transcription of the telomeric repeat-containing RNA (TERRA). In the absence of Bqt4, telomere shortening leads to deep increase in TERRA level and the concomitant formation of subtelomeric rearrangements (STEEx) that accumulate massively in quiescent cells. Taken together, our data demonstrate that Rap1-Bqt4-dependent telomere association to NE preserves telomere integrity in post-mitotic cells, preventing telomeric transcription and recombination. This defines the nuclear periphery as an area where recombination is restricted, creating a safe zone for telomeres of post-mitotic cells.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Membrana/genética , Membrana Nuclear/genética , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genética , Encurtamento do Telômero/genética , Proteínas de Ligação a Telômeros/genética , Divisão Celular/genética , Recombinação Genética , Schizosaccharomyces/genética , Complexo Shelterina , Telômero/genética , Transcrição GênicaRESUMO
Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.
Assuntos
Cromossomos Fúngicos/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Telômero/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase I/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Quadruplex G , Mutação , Proteína de Replicação A/genética , Proteínas de Schizosaccharomyces pombe/genética , Complexo Shelterina , Telômero/química , Encurtamento do Telômero , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Ploidy is stably maintained in most human somatic cells by a sequential and tight coordination of cell cycle events. Undesired whole genome doublings or duplications are frequent in tumours and have been quite recently described as macro-evolutionary events associated with poor prognosis. In vitro and in vivo studies suggest that polyploidy can favour genome instability, facilitate the formation and progression of tumours, and modify their sensitivity to chemotherapeutic agents. Stress is strongly related to changes in ploidy and whole genome doublings. In this review, we summarize different mechanisms that promote polyploidization, describe a new type of stress able to trigger WGDs in S. cerevisiae, histone stress, and provide some examples and theoretical scenarios that support that cancer cells might suffer from this type of stress. We finally highlight some results showing that the kinase Swe1 (Wee1 in humans) has a role in sensing histone levels before cells enter mitosis, thereby avoiding their undesired consequences on chromosome segregation and ploidy control.
Assuntos
Instabilidade Cromossômica , Histonas/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Estresse Fisiológico , Animais , Ciclo Celular , Suscetibilidade a Doenças , Instabilidade Genômica , Humanos , Poliploidia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The replication of the ends of linear chromosomes, or telomeres, poses unique problems, which must be solved to maintain genome integrity and to allow cell division to occur. Here, we describe and compare the timing and specific mechanisms that are required to initiate, control and coordinate synthesis of the leading and lagging strands at telomeres in yeasts, ciliates and mammals. Overall, it emerges that telomere replication relies on a strong synergy between the conventional replication machinery, telomere protection systems, DNA-damage-response pathways and chromosomal organization.
Assuntos
Telomerase/genética , Telômero/genética , Animais , Período de Replicação do DNA/genética , Humanos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
Telomere maintenance mechanism is poorly studied in quiescence, a reversible non-proliferative state. We previously described in fission yeast a new mode of repair of telomeres named STEEx, that specifically operates in post-mitotic cells harboring eroded telomeres. This mechanism, promoted by transcription-induced telomeric recombination, prevents cells to exit properly from quiescence, suggesting that STEEx act as an anti-proliferative barrier. Here, we further showed that STEEx are genetically controlled by the Tel1ATM- and Rad3ATR- dependent DDR pathways. We discussed the possibility that STEEx represent a boundary between quiescence and vegetative cycle.
Assuntos
Ciclo Celular/genética , Schizosaccharomyces/genética , Homeostase do Telômero/genética , Telômero/genética , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Mutação , Fosforilação , Proteínas Quinases/genética , Telomerase/genéticaRESUMO
Lagging-strand and leading-strand synthesis of chromosomes generates two structurally distinct ends at the telomeres. Based on sequence bias of yeast telomeres that contain a 250-300 bp array of C(1-3)A/ TG(1-3) repeats, we developed a method allowing us to distinguish which of the two daughter telomeres chromosome end-binding proteins bind to at the end of S phase. The single-stranded DNA-binding protein Cdc13 and the telomerase subunits Est1 and Est2 can bind to the two daughter telomeres, but only their binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex involved in both telomeric 5' nucleolytic resection and telomerase recruitment at short telomeres. Consistently, the MRX complex is mainly found to bind to the leading-strand telomere. Our results indicate that Cdc13 can bind to the telomeric template for lagging-strand replication. Since mre11-deficient strains have markedly short telomeres, telomere elongation by telomerase is likely to occur mainly at the leading-strand telomere.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/ultraestruturaRESUMO
Telomere protection in budding yeast requires the heterotrimer named CST (for Cdc13-Stn1-Ten1). Recent data show that CST components are conserved and required for telomere stability in a wide range of eukaryotes, even those utilizing the shelterin complex to protect their telomeres. A common function of these proteins might be to stimulate priming at the C-strand gap that remains after telomerase elongation, replication termination, and terminal processing. In light of the budding yeast situation, another conserved function of CST might well be the regulation of telomerase. The cohabitation at telomeres of CST and shelterin components highlights the complexity of telomere biology.
Assuntos
Ciclina B/metabolismo , Replicação do DNA/fisiologia , DNA Fúngico/metabolismo , Saccharomycetales/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Ciclina B/genética , DNA Fúngico/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Saccharomycetales/genética , Proteínas de Schizosaccharomyces pombe/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/genéticaRESUMO
The nuclear pore complex (NPC) is emerging as a center for recruitment of a class of "difficult to repair" lesions such as double-strand breaks without a repair template and eroded telomeres in telomerase-deficient cells. In addition to such pathological situations, a recent study by Su and colleagues shows that also physiological threats to genome integrity such as DNA secondary structure-forming triplet repeat sequences relocalize to the NPC during DNA replication. Mutants that fail to reposition the triplet repeat locus to the NPC cause repeat instability. Here, we review the types of DNA lesions that relocalize to the NPC, the putative mechanisms of relocalization, and the types of recombinational repair that are stimulated by the NPC, and present a model for NPC-facilitated repair.
Assuntos
DNA/genética , Poro Nuclear/genética , Reparo de DNA por Recombinação/genética , Dano ao DNA/genética , Replicação do DNA/genética , Humanos , Telômero/genética , Repetições de Trinucleotídeos/genéticaRESUMO
Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG1-3-like tracts present between subtelomeric X and Y' elements, which is followed by BIR-mediated non-reciprocal translocation of Y' element and terminal TG1-3 repeats from the donor end onto the shortened telomere. We found that choice of the Y' donor was not random, since both engineered telomere VII-L and native VI-R acquired Y' elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y' translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG1-3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y' translocation events taking place during presenescence are genetically separable from Rad51-dependent Y' amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y' translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.
Assuntos
Envelhecimento/genética , Proteínas de Ligação a DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Telômero/genética , Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Encurtamento do Telômero/genéticaRESUMO
In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.