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1.
Mol Cell ; 81(1): 183-197.e6, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33278361

RESUMEN

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.


Asunto(s)
Cromosomas Fúngicos/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
2.
J Biol Chem ; 300(3): 105708, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38311177

RESUMEN

A DNA double-strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination or nonhomologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of homologous recombination, and it is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harboring nuclease-dead dna2-1 compared with dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harboring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater resection defect compared with cells where DNA2 was deleted. Both the resection defect and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change; however, Nej1 increased as did the frequency of alt-end joining/microhomology-mediated end-joining repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Helicasas , Proteínas de Unión al ADN , Exodesoxirribonucleasas , Proteínas de Saccharomyces cerevisiae , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Genes Dev ; 30(8): 931-45, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-27056668

RESUMEN

High-resolution imaging shows that persistent DNA damage in budding yeast localizes in distinct perinuclear foci for repair. The signals that trigger DNA double-strand break (DSB) relocation or determine their destination are unknown. We show here that DSB relocation to the nuclear envelope depends on SUMOylation mediated by the E3 ligases Siz2 and Mms21. In G1, a polySUMOylation signal deposited coordinately by Mms21 and Siz2 recruits the SUMO targeted ubiquitin ligase Slx5/Slx8 to persistent breaks. Both Slx5 and Slx8 are necessary for damage relocation to nuclear pores. When targeted to an undamaged locus, however, Slx5 alone can mediate relocation in G1-phase cells, bypassing the requirement for polySUMOylation. In contrast, in S-phase cells, monoSUMOylation mediated by the Rtt107-stabilized SMC5/6-Mms21 E3 complex drives DSBs to the SUN domain protein Mps3 in a manner independent of Slx5. Slx5/Slx8 and binding to pores favor repair by ectopic break-induced replication and imprecise end-joining.


Asunto(s)
Roturas del ADN de Doble Cadena , Poro Nuclear/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sumoilación , Mutación , Membrana Nuclear/metabolismo , Unión Proteica , Fase S/fisiología , Saccharomyces cerevisiae/citología , Ubiquitina-Proteína Ligasas/metabolismo
4.
J Biol Chem ; 298(6): 101937, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35429499

RESUMEN

The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5'-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5'-resection mediated by the nuclease/helicase complex Dna2-Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11-Rad50-Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN/genética , ADN/metabolismo , Reparación del ADN/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
PLoS Genet ; 16(3): e1008422, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32187176

RESUMEN

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Carcinogénesis/genética , Saccharomyces cerevisiae/genética , Animales , Daño del ADN/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Genómica/métodos , Proteína Homóloga de MRE11/genética , Mutación/genética , Células Sf9 , Transducción de Señal/genética , Proteínas Supresoras de Tumor/genética
7.
Nat Chem Biol ; 16(1): 15-23, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31819272

RESUMEN

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.


Asunto(s)
Antineoplásicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de Unión al ARN/química , Sulfonamidas/farmacología , Secuencias de Aminoácidos , Calorimetría , Clonación Molecular , Fluorometría , Células HCT116 , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador , Péptidos y Proteínas de Señalización Intracelular/genética , Cinética , Proteínas Nucleares/metabolismo , Péptidos/química , Mutación Puntual , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteoma , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Ubiquitina-Proteína Ligasas/metabolismo
8.
Nucleic Acids Res ; 48(19): e111, 2020 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-33010172

RESUMEN

Ionizing radiation (IR) is environmentally prevalent and, depending on dose and linear energy transfer (LET), can elicit serious health effects by damaging DNA. Relative to low LET photon radiation (X-rays, gamma rays), higher LET particle radiation produces more disease causing, complex DNA damage that is substantially more challenging to resolve quickly or accurately. Despite the majority of human lifetime IR exposure involving long-term, repetitive, low doses of high LET alpha particles (e.g. radon gas inhalation), technological limitations to deliver alpha particles in the laboratory conveniently, repeatedly, over a prolonged period, in low doses and in an affordable, high-throughput manner have constrained DNA damage and repair research on this topic. To resolve this, we developed an inexpensive, high capacity, 96-well plate-compatible alpha particle irradiator capable of delivering adjustable, low mGy/s particle radiation doses in multiple model systems and on the benchtop of a standard laboratory. The system enables monitoring alpha particle effects on DNA damage repair and signalling, genome stability pathways, oxidative stress, cell cycle phase distribution, cell viability and clonogenic survival using numerous microscopy-based and physical techniques. Most importantly, this method is foundational for high-throughput genetic screening and small molecule testing in mammalian and yeast cells.


Asunto(s)
Partículas alfa/efectos adversos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Genética de Radiación/instrumentación , Células A549 , Ciclo Celular/efectos de la radiación , Células HeLa , Humanos , Estrés Oxidativo/efectos de la radiación , Saccharomyces cerevisiae , Transducción de Señal/efectos de la radiación
9.
Mol Cell ; 48(1): 98-108, 2012 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-22885006

RESUMEN

The cohesin complex holds together newly replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins, and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on γH2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50Δ mutants and in cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a function for cohesin in the maintenance of genome integrity during S phase.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Genes Fúngicos , Histonas/metabolismo , Hidroxiurea/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico , Cohesinas
10.
PLoS Genet ; 12(8): e1006268, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27564449

RESUMEN

SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). SIR4 recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, nse3-1/ sir4 Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation.


Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Telómero/genética , Transcripción Genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sumoilación/genética , Proteínas de Unión a Telómeros/genética , Cohesinas
11.
J Biol Chem ; 292(35): 14576-14586, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28679532

RESUMEN

Double strand breaks (DSBs) represent highly deleterious DNA damage and need to be accurately repaired. Homology-directed repair and non-homologous end joining (NHEJ) are the two major DSB repair pathways that are highly conserved from yeast to mammals. The choice between these pathways is largely based on 5' to 3' DNA resection, and NHEJ proceeds only if resection has not been initiated. In yeast, yKu70/80 rapidly localizes to the break, protecting DNA ends from nuclease accessibility, and recruits additional NHEJ factors, including Nej1 and Lif1. Cells harboring the nej1-V338A mutant exhibit NHEJ-mediated repair deficiencies and hyper-resection 0.15 kb from the DSB that was dependent on the nuclease activity of Dna2-Sgs1. The integrity of Nej1 is also important for inhibiting long-range resection, 4.8 kb from the break, and for preventing the formation of large genomic deletions at sizes >700 bp around the break. Nej1V338A localized to a DSB similarly to WT Nej1, indicating that the Nej1-Lif1 interaction becomes critical for blocking hyper-resection mainly after their recruitment to the DSB. This work highlights that Nej1 inhibits 5' DNA hyper-resection mediated by Dna2-Sgs1, a function distinct from its previously reported role in supporting Dnl4 ligase activity, and has implications for repair pathway choice and resection regulation upon DSB formation.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sustitución de Aminoácidos , ADN Helicasas/química , ADN Helicasas/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Eliminación de Gen , Viabilidad Microbiana , Mutación Puntual , Multimerización de Proteína , Transporte de Proteínas , RecQ Helicasas/química , RecQ Helicasas/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidad de la Especie
12.
Biochem Cell Biol ; 94(5): 433-440, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27604033

RESUMEN

The nucleus is a hub for gene expression and is a highly organized entity. The nucleoplasm is heterogeneous, owing to the preferential localization of specific metabolic factors, which lead to the definition of nuclear compartments or bodies. The genome is organized into chromosome territories, as well as heterochromatin and euchromatin domains. Recent observations have indicated that nuclear organization is important for maintaining genomic stability. For example, nuclear organization has been implicated in stabilizing damaged DNA, repair-pathway choice, and in preventing chromosomal rearrangements. Over the past decade, several studies have revealed that dynamic changes in the nuclear architecture are important during double-strand break repair. Stemming from work in yeast, relocation of a damaged site prior to repair appears to be at least partially conserved in multicellular eukaryotes. In this review, we will discuss genome and nucleoplasm architecture, particularly the importance of the nuclear periphery in genome stability. We will also discuss how the site of relocation regulates repair-pathway choice.


Asunto(s)
Núcleo Celular/química , Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos
13.
Bioconjug Chem ; 26(8): 1839-49, 2015 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-26230938

RESUMEN

We have recently described a method for tyrosine-ligation of complex glycans that was proven efficient for the site selective coupling of GBS capsular polysaccharides (PSs). Herein, we explored the effect of conjugation of type V polysaccharide onto predetermined lysine or tyrosine residues of the GBS67 pilus protein with the dual role of T-cell carrier for the PS and antigen. For the preparation of a conjugate at predetermined lysine residues of the protein, we investigated a two-step procedure based on microbial Transglutaminase (mTGase) catalyzed insertion of a tag bearing an azide for following copper-free strain-promoted azide-alkyne [3 + 2] cycloaddition (SPAAC) with the polysaccharide. Two glycoconjugates were obtained by tyrosine-ligation through the known SPAAC and a novel thiol-maleimide addition based approach. Controls were prepared by random conjugation of PSV to GBS67 and CRM197, a carrier protein present in many commercial vaccines. Immunological evaluation in mice showed that all the site-directed constructs were able to induce good levels of anti-polysaccharide and anti-protein antibodies inducing osponophagocytic killing of strains expressing individually PSV or GBS67. GBS67 randomly conjugated to PSV showed carrier properties similar to CRM197. Among the tested site-directed conjugates, tyrosine-directed ligation and thiol-malemide addition was elected as the best combination to ensure production of anti-polysaccharide and anti-protein functional antibodies (in vitro opsonophagocytic killing titers) comparable to the controls made by random conjugation, while avoiding anti-linker antibodies. Our findings demonstrate that (i) mTGase based conjugation at lysine residues is an alternative approach for the synthesis of large capsular polysaccharide-protein conjugates; (ii) GBS67 can be used with the dual role of antigen and carrier for PSV; and (iii) thiol-maleimide addition in combination with tyrosine-ligation ensures the production of anti-polysaccharide and anti-protein functional antibodies while maintaining low levels of anti-linker antibodies. Site-specific conjugation methods aid in defining conjugation site and chemistry in carbohydrate-protein conjugates.


Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/farmacología , Glicoconjugados/farmacología , Polisacáridos/inmunología , Infecciones Estreptocócicas/prevención & control , Streptococcus/inmunología , Vacunas Conjugadas/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Vacunas Bacterianas/inmunología , Secuencia de Carbohidratos , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicoconjugados/inmunología , Inmunización , Ratones , Datos de Secuencia Molecular , Polisacáridos/química , Infecciones Estreptocócicas/inmunología , Tirosina/química , Tirosina/inmunología , Vacunas Conjugadas/inmunología
14.
Angew Chem Int Ed Engl ; 54(45): 13198-203, 2015 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-26350581

RESUMEN

A series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O-antigen to different amino acids of CRM197 protein carrier. In particular, two novel methods for site-selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH-controlled transglutaminase-catalyzed modification of lysine, respectively. Importantly, conjugation at the C186-201 bond resulted in significantly higher anti O-antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies.


Asunto(s)
Glicoconjugados/química , Glicoconjugados/inmunología , Antígenos O/química , Antígenos O/inmunología , Vacunas contra la Salmonella/química , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Conformación Molecular , Salmonella typhimurium/química
15.
FEBS Lett ; 598(2): 187-198, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38058218

RESUMEN

Nucleoplasmin (NPM) histone chaperones regulate distinct processes in the nucleus and nucleolus. While intrinsically disordered regions (IDRs) are hallmarks of NPMs, it is not clear whether all NPM functions require these unstructured features. We assessed the importance of IDRs in a yeast NPM-like protein and found that regulation of rDNA copy number and genetic interactions with the nucleolar RNA surveillance machinery require the highly conserved FKBP prolyl isomerase domain, but not the NPM domain or IDRs. By contrast, transcriptional repression in the nucleus requires IDRs. Furthermore, multiple lysines in polyacidic serine/lysine motifs of IDRs are required for both lysine polyphosphorylation and NPM-mediated transcriptional repression. These results demonstrate that this NPM-like protein relies on IDRs only for some of its chromatin-related functions.


Asunto(s)
Chaperonas de Histonas , Lisina , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Nucleoplasminas/metabolismo , Lisina/metabolismo , Cromatina/genética , Cromatina/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
16.
Science ; 385(6704): 91-99, 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-38963839

RESUMEN

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.


Asunto(s)
Anemia de Células Falciformes , Hemoglobina Fetal , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Animales , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Humanos , Ratones , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteolisis , Macaca fascicularis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Cristalografía por Rayos X
17.
J Biol Chem ; 287(14): 11374-83, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22303010

RESUMEN

The Smc5/6 complex belongs to the SMC (structural maintenance of chromosomes) family, which also includes cohesin and condensin. In Saccharomyces cerevisiae, the Smc5/6 complex contains six essential non-Smc elements, Nse1-6. Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 small ubiquitin-like modifier (SUMO) ligase important for Smc5 sumoylation. Characterization of two temperature-sensitive mutants, nse5-ts1 and nse5-ts2, demonstrated the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, formation of fork-associated homologous recombination intermediates, and hydroxyurea sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, and for its functionality during replication stress.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , ADN de Hongos/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Alelos , Proteínas Cromosómicas no Histona/genética , Replicación del ADN/efectos de los fármacos , ADN de Hongos/química , Hidroxiurea/farmacología , Mutación , Fenotipo , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico/efectos de los fármacos , Sumoilación/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo
18.
EMBO J ; 28(8): 1142-56, 2009 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-19279665

RESUMEN

The Mre11-Rad50-Xrs2 (MRX) complex has an important function in the maintenance of genomic integrity by contributing to the detection and repair of chromosome breaks. Here we show that the complex is recruited to sites of paused forks where it stabilizes the association of essential replisome components. Interestingly, this function is not dependent on the S phase checkpoint or the nuclease activity of Mre11. We find that disruption of the MRX complex leads to a loss of fork recovery and a failure to properly complete DNA replication when cells are exposed to replication stress. Our data suggest that one critical function of the MRX complex during replication is to promote the cohesion of sister chromatids at paused forks, offering an explanation for why MRX deficiency leads to a loss of cell viability and high levels of chromosome rearrangements under conditions of replication stress.


Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Complejos Multiproteicos/metabolismo , Fase S/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , ADN Polimerasa II/metabolismo , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Epistasis Genética , Exodesoxirribonucleasas/genética , Humanos , Hidroxiurea/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
19.
J Cell Biol ; 222(7)2023 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-37042812

RESUMEN

The nuclear envelope (NE) is important in maintaining genome organization. The role of lipids in communication between the NE and telomere regulation was investigated, including how changes in lipid composition impact gene expression and overall nuclear architecture. Yeast was treated with the non-metabolizable lysophosphatidylcholine analog edelfosine, known to accumulate at the perinuclear ER. Edelfosine induced NE deformation and disrupted telomere clustering but not anchoring. Additionally, the association of Sir4 at telomeres decreased. RNA-seq analysis showed altered expression of Sir-dependent genes located at sub-telomeric (0-10 kb) regions, consistent with Sir4 dispersion. Transcriptomic analysis revealed that two lipid metabolic circuits were activated in response to edelfosine, one mediated by the membrane sensing transcription factors, Spt23/Mga2, and the other by a transcriptional repressor, Opi1. Activation of these transcriptional programs resulted in higher levels of unsaturated fatty acids and the formation of nuclear lipid droplets. Interestingly, cells lacking Sir proteins displayed resistance to unsaturated-fatty acids and edelfosine, and this phenotype was connected to Rap1.


Asunto(s)
Membrana Nuclear , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero , Proteínas de la Membrana/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Éteres Fosfolípidos/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Telómero/genética , Telómero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
20.
Cell Chem Biol ; 30(3): 235-247.e12, 2023 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-36863346

RESUMEN

Malignant tumors can evade destruction by the immune system by attracting immune-suppressive regulatory T cells (Treg) cells. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Treg cells, and IKZF2 deficiency reduces tumor growth in mice. Here we report the discovery of NVP-DKY709, a selective molecular glue degrader of IKZF2 that spares IKZF1/3. We describe the recruitment-guided medicinal chemistry campaign leading to NVP-DKY709 that redirected the degradation selectivity of cereblon (CRBN) binders from IKZF1 toward IKZF2. Selectivity of NVP-DKY709 for IKZF2 was rationalized by analyzing the DDB1:CRBN:NVP-DKY709:IKZF2(ZF2 or ZF2-3) ternary complex X-ray structures. Exposure to NVP-DKY709 reduced the suppressive activity of human Treg cells and rescued cytokine production in exhausted T-effector cells. In vivo, treatment with NVP-DKY709 delayed tumor growth in mice with a humanized immune system and enhanced immunization responses in cynomolgus monkeys. NVP-DKY709 is being investigated in the clinic as an immune-enhancing agent for cancer immunotherapy.


Asunto(s)
Neoplasias , Factores de Transcripción , Animales , Humanos , Ratones , Factor de Transcripción Ikaros , Inmunoterapia , Neoplasias/terapia , Neoplasias/metabolismo , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/metabolismo
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