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2.
PLoS Genet ; 19(2): e1010639, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36749784

RESUMEN

The bypass of DNA lesions that block replicative polymerases during DNA replication relies on DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway depends on specialized DNA polymerases that incorporate nucleotides in front of base lesions, potentially inducing mutagenesis. Two error-free pathways can bypass the lesions: the template switching pathway, which uses the sister chromatid as a template, and the homologous recombination pathway (HR), which also can use the homologous chromosome as template. The balance between error-prone and error-free pathways controls the mutagenesis level. Therefore, it is crucial to precisely characterize factors that influence the pathway choice to better understand genetic stability at replication forks. In yeast, the complex formed by the Rad51 paralogs Rad55 and Rad57 promotes HR and template-switching at stalled replication forks. At DNA double-strand breaks (DSBs), this complex promotes Rad51 filament formation and stability, notably by counteracting the Srs2 anti-recombinase. To explore the role of the Rad55-Rad57 complex in error-free pathways, we monitored the genetic interactions between Rad55-Rad57, the translesion polymerases Polζ or Polη, and Srs2 following UV radiation that induces mostly single-strand DNA gaps. We found that the Rad55-Rad57 complex was involved in three ways. First, it protects Rad51 filaments from Srs2, as it does at DSBs. Second, it promotes Rad51 filament stability independently of Srs2. Finally, we observed that UV-induced HR is almost abolished in Rad55-Rad57 deficient cells, and is partially restored upon Polζ or Polη depletion. Hence, we propose that the Rad55-Rad57 complex is essential to promote Rad51 filament stability on single-strand DNA gaps, notably to counteract the error-prone TLS polymerases and mutagenesis.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/genética , ADN/metabolismo , Daño del ADN/genética , ADN Helicasas/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Rayos Ultravioleta
3.
Trends Genet ; 38(7): 641-645, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35397934

RESUMEN

2021 marked the 80th anniversary of Barbara McClintock's pioneering article on the breakage-fusion-bridge (BFB) cycle. Of the three steps of the BFB cycle, breakage remains the least understood despite its major contribution to mutagenesis. We discuss recent findings shedding light on how chromatin bridges break in yeast and animal cells.


Asunto(s)
Ciclo Celular/genética , Cromosomas , Citogenética/historia , Saccharomyces cerevisiae , Inestabilidad Genómica , Historia del Siglo XX , Saccharomyces cerevisiae/genética , Telómero
4.
EMBO J ; 41(1): e108813, 2022 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-34817085

RESUMEN

Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In the yeast Saccharomyces cerevisiae, the silent information regulator (SIR) complex assembles heterochromatin-like chromatin at sub-telomeric chromosomal regions. SIR-mediated repressive chromatin limits DNA double-strand break (DSB) resection, thus protecting damaged chromosome ends during homologous recombination (HR). As resection initiation represents the crossroads between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR-mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Via physical interaction with the Sae2 protein, Sir3 impairs Sae2-dependent functions of the MRX (Mre11-Rad50-Xrs2) complex, thereby limiting Mre11-mediated resection, delaying MRX removal from DSB ends, and promoting NHEJ.


Asunto(s)
Reparación del ADN por Unión de Extremidades , Endonucleasas/metabolismo , Heterocromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Endonucleasas/química , Mutación Puntual/genética , Unión Proteica , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Telómero/metabolismo
5.
Nucleic Acids Res ; 49(12): 6817-6831, 2021 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-34125900

RESUMEN

Chromosome fusions threaten genome integrity and promote cancer by engaging catastrophic mutational processes, namely chromosome breakage-fusion-bridge cycles and chromothripsis. Chromosome fusions are frequent in cells incurring telomere dysfunctions or those exposed to DNA breakage. Their occurrence and therefore their contribution to genome instability in unchallenged cells is unknown. To address this issue, we constructed a genetic assay able to capture and quantify rare chromosome fusions in budding yeast. This chromosome fusion capture (CFC) assay relies on the controlled inactivation of one centromere to rescue unstable dicentric chromosome fusions. It is sensitive enough to quantify the basal rate of end-to-end chromosome fusions occurring in wild-type cells. These fusions depend on canonical nonhomologous end joining (NHEJ). Our results show that chromosome end protection results from a trade-off at telomeres between positive effectors (Rif2, Sir4, telomerase) and a negative effector partially antagonizing them (Rif1). The CFC assay also captures NHEJ-dependent chromosome fusions induced by ionizing radiation. It provides evidence for chromosomal rearrangements stemming from a single photon-matter interaction.


Asunto(s)
Aberraciones Cromosómicas , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Telómero , Centrómero , Técnicas Genéticas , Radiación Ionizante , Saccharomyces cerevisiae/genética , Telómero/metabolismo , Homeostasis del Telómero
6.
Nat Commun ; 12(1): 2763, 2021 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-33980827

RESUMEN

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencias de Aminoácidos , Cromosomas Fúngicos/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/química , Exodesoxirribonucleasas/química , Modelos Moleculares , Complejos Multiproteicos , Mutación , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genética
7.
Mol Cell ; 75(1): 131-144.e3, 2019 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-31204167

RESUMEN

In Saccharomyces cerevisiae, dicentric chromosomes stemming from telomere fusions preferentially break at the fusion. This process restores a normal karyotype and protects chromosomes from the detrimental consequences of accidental fusions. Here, we address the molecular basis of this rescue pathway. We observe that tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. We also show that condensins generate forces sufficient to rapidly refold dicentrics prior to breakage by cytokinesis and are essential to the preferential breakage at telomere fusions. Thus, the rescue of fused telomeres results from a condensin- and Rap1-driven chromosome folding that favors fusion entrapment where abscission takes place. Because a close spacing between the DNA-bound Rap1 molecules is essential to this process, Rap1 may act by stalling condensins.


Asunto(s)
Adenosina Trifosfatasas/genética , Cromosomas Fúngicos/metabolismo , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Telómero/metabolismo , Factores de Transcripción/genética , Adenosina Trifosfatasas/metabolismo , Puntos de Rotura del Cromosoma , Cromosomas Fúngicos/ultraestructura , Citocinesis/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Cariotipo , Modelos Genéticos , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Telómero/ultraestructura , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismo
8.
Genome Biol Evol ; 11(2): 572-585, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30668669

RESUMEN

Sir4 is a core component of heterochromatin found in yeasts of the Saccharomycetaceae family, whose general hallmark is to harbor a three-loci mating-type system with two silent loci. However, a large part of the Sir4 amino acid sequences has remained unexplored, belonging to the dark proteome. Here, we analyzed the phylogenetic profile of yet undescribed foldable regions present in Sir4 as well as in Esc1, an Sir4-interacting perinuclear anchoring protein. Within Sir4, we identified a new conserved motif (TOC) adjacent to the N-terminal KU-binding motif. We also found that the Esc1-interacting region of Sir4 is a Dbf4-related H-BRCT domain, only present in species possessing the HO endonuclease and in Kluveryomyces lactis. In addition, we found new motifs within Esc1 including a motif (Esc1-F) that is unique to species where Sir4 possesses an H-BRCT domain. Mutagenesis of conserved amino acids of the Sir4 H-BRCT domain, known to play a critical role in the Dbf4 function, shows that the function of this domain is separable from the essential role of Sir4 in transcriptional silencing and the protection from HO-induced cutting in Saccharomyces cerevisiae. In the more distant methylotrophic clade of yeasts, which often harbor a two-loci mating-type system with one silent locus, we also found a yet undescribed H-BRCT domain in a distinct protein, the ISWI2 chromatin-remodeling factor subunit Itc1. This study provides new insights on yeast heterochromatin evolution and emphasizes the interest of using sensitive methods of sequence analysis for identifying hitherto ignored functional regions within the dark proteome.


Asunto(s)
Evolución Molecular , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Secuencia Conservada , Heterocromatina , Dominios Proteicos/genética , Saccharomyces cerevisiae
9.
Genes Dev ; 29(3): 322-36, 2015 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25644606

RESUMEN

Dicentric chromosomes are unstable products of erroneous DNA repair events that can lead to further genome rearrangements and extended gene copy number variations. During mitosis, they form anaphase bridges, resulting in chromosome breakage by an unknown mechanism. In budding yeast, dicentrics generated by telomere fusion break at the fusion, a process that restores the parental karyotype and protects cells from rare accidental telomere fusion. Here, we observed that dicentrics lacking telomere fusion preferentially break within a 25- to 30-kb-long region next to the centromeres. In all cases, dicentric breakage requires anaphase exit, ruling out stretching by the elongated mitotic spindle as the cause of breakage. Instead, breakage requires cytokinesis. In the presence of dicentrics, the cytokinetic septa pinch the nucleus, suggesting that dicentrics are severed after actomyosin ring contraction. At this time, centromeres and spindle pole bodies relocate to the bud neck, explaining how cytokinesis can sever dicentrics near centromeres.


Asunto(s)
Centrómero/genética , Rotura Cromosómica , Cromosomas Fúngicos/genética , Citocinesis , Saccharomyces cerevisiae/genética , Telómero/metabolismo , División del Núcleo Celular , Mitosis
10.
Mol Cell Oncol ; 1(3): e963438, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-27308342

RESUMEN

The telomeres of eukaryotes are stable open double-strand ends that coexist with nonhomologous end joining (NHEJ), the repair pathway that directly ligates DNA ends generated by double-strand breaks. Since a single end-joining event between 2 telomeres generates a circular chromosome or an unstable dicentric chromosome, NHEJ must be prevented from acting on telomeres. Multiple mechanisms mediated by telomere factors act in synergy to achieve this inhibition.

11.
EMBO J ; 32(6): 805-15, 2013 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-23417015

RESUMEN

In eukaryotes, permanent inhibition of the non-homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2-related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates. We propose that one of Uls1 functions is to clear non-functional poly-SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly-SUMOylated proteins on DNA in eukaryotes.


Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Helicasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Telómero/metabolismo , ADN Helicasas/metabolismo , Regulación hacia Abajo , Organismos Modificados Genéticamente , Peptidil Transferasas/metabolismo , Peptidil Transferasas/fisiología , Unión Proteica , Multimerización de Proteína/fisiología , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Sumoilación/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Proteínas de Unión al GTP rap1/metabolismo
12.
Genes Dev ; 24(7): 720-33, 2010 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20360388

RESUMEN

Nonhomologous end-joining (NHEJ) inhibition at telomeres ensures that native chromosome ends do not fuse together. But the occurrence and consequences of rare telomere fusions are not well understood. It is notably unclear whether a telomere fusion could be processed to restore telomere ends. Here we address the behavior of individual dicentrics formed by telomere fusion in the yeast Saccharomyces cerevisiae. Our approach was to first stabilize and amplify fusions between two chromosomes by temporarily inactivating one centromere. Next we analyzed dicentric breakage following centromere reactivation. Unexpectedly, dicentrics often break at the telomere fusions during progression through mitosis, a process that restores the parental chromosomes. This unforeseen result suggests a rescue pathway able to process telomere fusions and to back up NHEJ inhibition at telomeres.


Asunto(s)
Rotura Cromosómica , Cromosomas Fúngicos/genética , Saccharomyces cerevisiae/genética , Telómero/genética , Centrómero/genética , Mitosis/genética
13.
Genes Dev ; 22(9): 1153-8, 2008 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-18451106

RESUMEN

The nonhomologous end-joining (NHEJ) repair pathway is inhibited at telomeres, preventing chromosome fusion. In budding yeast Saccharomyces cerevisiae, the Rap1 protein directly binds the telomere sequences and is required for NHEJ inhibition. Here we show that the Rap1 C-terminal domain establishes two parallel inhibitory pathways through the proteins Rif2 and Sir4. In addition, the central domain of Rap1 inhibits NHEJ independently of Rif2 and Sir4. Thus, Rap1 establishes several independent pathways to prevent telomere fusions. We discuss a possible mechanism that would explain Rif2 multifunctionality at telomeres and the recent evolutionary origin of Rif2 from an origin recognition complex (ORC) subunit.


Asunto(s)
Reparación del ADN , Recombinación Genética/genética , Transducción de Señal , Telómero/genética , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Complejo Shelterina , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genética
14.
Genetics ; 172(4): 2689-94, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16452137

RESUMEN

In yeast, the nonhomologous end joining pathway (NHEJ) mobilizes the DNA polymerase Pol4 to repair DNA double-strand breaks when gap filling is required prior to ligation. Using telomere-telomere fusions caused by loss of the telomeric protein Rap1 and double-strand break repair on transformed DNA as assays for NHEJ between fully uncohesive ends, we show that Pol4 is able to extend a 3'-end whose last bases are mismatched, i.e., mispaired or unpaired, to the template strand.


Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/fisiología , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genética , Alelos , Disparidad de Par Base , Secuencia de Bases , ADN Polimerasa beta , Cartilla de ADN , Reparación del ADN , Datos de Secuencia Molecular , Plásmidos/metabolismo , Complejo Shelterina
15.
EMBO J ; 24(17): 3117-27, 2005 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-16096640

RESUMEN

Telomeres protect chromosomes from end-to-end fusions. In yeast Saccharomyces cerevisiae, the protein Rap1 directly binds telomeric DNA. Here, we use a new conditional allele of RAP1 and show that Rap1 loss results in frequent fusions between telomeres. Analysis of the fusion point with restriction enzymes indicates that fusions occur between telomeres of near wild-type length. Telomere fusions are not observed in cells lacking factors required for nonhomologous end joining (NHEJ), including Lig4 (ligase IV), KU and the Mre11 complex. SAE2 and TEL1 do not affect the frequency of fusions. Together, these results show that Rap1 is essential to block NHEJ between telomeres. Since the presence of Rap1 at telomeres has been conserved through evolution, the establishment of NHEJ suppression by Rap1 could be universal.


Asunto(s)
Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Proteínas de Unión a Telómeros/fisiología , Telómero/fisiología , Factores de Transcripción/fisiología , Supervivencia Celular/fisiología , Cromosomas Fúngicos/genética , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , Reparación del ADN , Enzimas de Restricción del ADN/química , Endodesoxirribonucleasas/metabolismo , Endonucleasas , Exodesoxirribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas , Recombinación Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/química , Factores de Transcripción/genética
16.
Mol Cell ; 10(5): 1189-99, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12453425

RESUMEN

The stability of DNA ends generated by the HO endonuclease in yeast is surprisingly high with a half-life of more than an hour. This transient stability is unaffected by mutations that abolish nonhomologous end joining (NHEJ). The unprocessed ends interact with Yku70p and Yku80p, two proteins required for NHEJ, but not significantly with Rad52p, a protein involved in homologous recombination (HR). Repair of a double-strand break by NHEJ is unaffected by the possibility of HR, although the use of HR is increased in NHEJ-defective cells. Partial in vitro 5' strand processing suppresses NHEJ but not HR. These results show that NHEJ precedes HR temporally, and that the availability of substrate dictates the particular pathway used. We propose that transient stability of DNA ends is a foundation for the permanent stability of telomeres.


Asunto(s)
Reparación del ADN , ADN/química , Conformación de Ácido Nucleico , Recombinación Genética , Unión Competitiva , ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Modelos Genéticos , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Unión Proteica , Proteínas de Saccharomyces cerevisiae , Telómero/metabolismo , Factores de Tiempo , Levaduras/metabolismo
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