Accumulation of exoglucanase activity in yeast secretory mutants blocked at the endoplasmic reticulum level

AbstractSaccharomyces cerevisiae HMSF-176 (sec 18), a thermosensitive secretory mutant blocked at the endoplasmic reticulum (ER) level, drastically increased its osmotic sensitivity when grown at the restrictive temperature of 37°C in high glucose concentration. This fact led to the erroneous interpretation that glucanases were inactive when localized in the ER. The development of a suitable osmotic stabilizer now indicates that sec 18 accumulates exoglucanase activity. Another ER-blocked mutant behaved in a similar way. All the accumulated exoglucanase was found in a soluble form. By contrast, a significant portion of the accumulated invertase remained in a membrane-bound form

Rad51-Rad52 mediated maintenance of centromeric chromatin in candida albicans

La ubicación específica de un centrómero en la mayoría de los eucariotas no depende únicamente de la secuencia de ADN. Sin embargo, los determinantes no genéticos de identidad de un centrómero no están claramente definidos. Aunque varios mecanismos, de forma individual o en conjunto, pueden especificar centrómeros epigenéticos, la mayoría de los estudios en este área se centran en un factor universal, un centromerospecific histona H3 variante CENP-A, a menudo considerado como el determinante de la identidad epigenética del centrómero. A pesar de la sincronización variable de su carga en centrómeros través de las especies, una replicación junto a una deposición en fase temprana S de CENP-A se encuentra en la mayoría de los centrómeros de levadura. Centrómeros son las regiones más tempranas de replicación cromosómica en una levadura en ciernes patógeno Candida albicans. Al aplicar un ensayo de electroforesis en gel de agarosa de dos dimensiones se identifican los orígenes de replicación (ORI7-LI y ORI7-RI) proxima a un centrómero temprano replicante (CEN7) en C. albicans. Se demuestra que las horquillas de replicación se estancan en CEN7 de una manera dependiente del cinetocoro y el estancamiento tenedor se reduce en ausencia de la recombinación homóloga (HR) proteínas Rad51 y Rad52. La supresión de ORI7-RI provoca una reducción significativa en la señal de tenedor estancado y una mayor tasa de pérdida del cromosoma alterado 7. Las proteínas de recursos humanos, Rad51 y Rad52, han demostrado que desempeñan un papel en el reinicio del tenedor. La microscopía confocal muestra cinetocoros declustered en rad51 y rad52 mutantes, que son evidencia de la disrupción cinetocoro. Los niveles de CENP-ACaCse4 en centrómeros, como se determina por los experimentos de inmunoprecipitación de la cromatina (ChIP), se reducen en ausencia de Rad51 / Rad52 que resulta en la interrupción de la estructura cinetocoro. Además, el análisis de transferencia Western revela que las moléculas de CENP-A deslocalizados en mutantes de recursos humanos se degradan de un modo similar como en otros mutantes kinetochore descritos antes. Finalmente, los ensayos de co-inmunoprecipitación indican que Rad51 y Rad52 interactuan físicamente con CENPA CaCse4 in vivo. Por lo tanto, las proteínas Rad51 y Rad52 de recursos epigenéticos humanos mantienen el funcionamiento del centrómero mediante la regulación de los niveles de CENPA CaCse4 en los sitios de parada programados en los principios centrómeros a replicar.Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromerespecific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENPA CaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENPA CaCse4 levels at the programmed stall sites of early replicating centromeresK. Sanyal y D. D. Dubey han recibido financiación del Government of India. Department of Biotechnology S. Mitra fue Senior Research Fellowship financiado por Council of Scientific and Industrial Research K Sanyal recibió ayuda de Jawaharlal Nehru Centre for Advanced Scientific Research G. Larriba fue financiado por Junta de Extremadura, Ayuda a grupos CCV014, Fondos FEDER; y por el Ministerio de Ciencia e Innovación, SAF2010-19848peerReviewe

Role of homologous recombination genes in repair of alkylation base damage by Candida albicans

Los mutantes de Candida albicans deficientes en recombinación homóloga (HR) son extremadamente sensibles al agente alquilante metil-metano-sulfonato (MMS). Aquí hemos investigado el papel de los genes HR en la protección y reparación de los cromosomas de C. albicans aprovechando la propiedad calor-lábil (55 °C) del daño de la base inducido por MMS. Los tratamientos agudos de MMS de las células cíclicas causaron fragmentación de cromosomas in vitro (55 °C) debido a la generación de roturas dependientes del calor (HDB), pero no in vivo (30 °C). Tras la eliminación del MMS de tipo silvestre, las células recuperaron la escalera cromosómica independientemente de que se transfirieran a extracto de levadura/peptona/dextrosa (YPD) o a solución salina amortiguadora de fosfatos (PBS); sin embargo, la reparación de la restitución de HDB/cromosomas fue más rápida en la YPD, lo que sugiere que se aceleró por la energía metabólica y se alimentó aún más por el subsiguiente crecimiento excesivo de los supervivientes. En comparación con el tipo silvestre CAI4, la restitución de cromosomas en la EPJ no se alteró en un derivado isogénico Carad59, mientras que se retrasó considerablemente en sus homólogos Carad51 y Carad52. Sin embargo, cuando se produjo la incubación posterior al MMS en la SAF, la restitución de cromosomas en los mutantes de tipo silvestre y de RH se produjo con una cinética similar, lo que sugiere que la exquisita sensibilidad de los mutantes Carad51 y Carad52 al MMS se debe a un reinicio defectuoso de la horquilla. En general, nuestros resultados demuestran que la reparación de los HDB por células en reposo de C. albicans es bastante independiente de CaRad51, CaRad52 y CaRad59, lo que sugiere que se produce principalmente por reparación de escisión de la base (BER).Candida albicans mutants deficient in homologous recombination (HR) are extremely sensitive to the alkylating agent methyl-methane-sulfonate (MMS). Here, we have investigated the role of HR genes in the protection and repair of C. albicans chromosomes by taking advantage of the heat-labile property (55 °C) of MMS-induced base damage. Acute MMS treatments of cycling cells caused chromosome fragmentation in vitro (55 °C) due to the generation of heat-dependent breaks (HDBs), but not in vivo (30 °C). Following removal of MMS wild type, cells regained the chromosome ladder regardless of whether they were transferred to yeast extract/peptone/dextrose (YPD) or to phosphate buffer saline (PBS); however, repair of HDB/chromosome restitution was faster in YPD, suggesting that it was accelerated by metabolic energy and further fueled by the subsequent overgrowth of survivors. Compared to wild type CAI4, chromosome restitution in YPD was not altered in a Carad59 isogenic derivative, whereas it was significantly delayed in Carad51 and Carad52 counterparts. However, when post-MMS incubation took place in PBS, chromosome restitution in wild type and HR mutants occurred with similar kinetics, suggesting that the exquisite sensitivity of Carad51 and Carad52 mutants to MMS is due to defective fork restart. Overall, our results demonstrate that repair of HDBs by resting cells of C. albicans is rather independent of CaRad51, CaRad52, and CaRad59, suggesting that it occurs mainly by base excision repair (BER).peerReviewe

The Candida albicans Ku70 Modulates Telomere Length and Structure by Regulating Both Telomerase and Recombination

The heterodimeric Ku complex has been shown to participate in DNA repair and telomere regulation in a variety of organisms. Here we report a detailed characterization of the function of Ku70 in the diploid fungal pathogen Candida albicans. Both ku70 heterozygous and homozygous deletion mutants have a wild-type colony and cellular morphology, and are not sensitive to MMS or UV light. Interestingly, we observed complex effects of KU70 gene dosage on telomere lengths, with the KU70/ku70 heterozygotes exhibiting slightly shorter telomeres, and the ku70 null strain exhibiting long and heterogeneous telomeres. Analysis of combination mutants suggests that the telomere elongation in the ku70 null mutant is due mostly to unregulated telomerase action. In addition, elevated levels of extrachromosomal telomeric circles were detected in the null mutant, consistent with activation of aberrant telomeric recombination. Altogether, our observations point to multiple mechanisms of the Ku complex in telomerase regulation and telomere protection in C. albicans, and reveal interesting similarities and differences in the mechanisms of the Ku complex in disparate systems

Role of Homologous Recombination Genes in Repair of Alkylation Base Damage by Candida albicans

Candida albicans mutants deficient in homologous recombination (HR) are extremely sensitive to the alkylating agent methyl-methane-sulfonate (MMS). Here, we have investigated the role of HR genes in the protection and repair of C. albicans chromosomes by taking advantage of the heat-labile property (55 °C) of MMS-induced base damage. Acute MMS treatments of cycling cells caused chromosome fragmentation in vitro (55 °C) due to the generation of heat-dependent breaks (HDBs), but not in vivo (30 °C). Following removal of MMS wild type, cells regained the chromosome ladder regardless of whether they were transferred to yeast extract/peptone/dextrose (YPD) or to phosphate buffer saline (PBS); however, repair of HDB/chromosome restitution was faster in YPD, suggesting that it was accelerated by metabolic energy and further fueled by the subsequent overgrowth of survivors. Compared to wild type CAI4, chromosome restitution in YPD was not altered in a Carad59 isogenic derivative, whereas it was significantly delayed in Carad51 and Carad52 counterparts. However, when post-MMS incubation took place in PBS, chromosome restitution in wild type and HR mutants occurred with similar kinetics, suggesting that the exquisite sensitivity of Carad51 and Carad52 mutants to MMS is due to defective fork restart. Overall, our results demonstrate that repair of HDBs by resting cells of C. albicans is rather independent of CaRad51, CaRad52, and CaRad59, suggesting that it occurs mainly by base excision repair (BER)

Partner Choice in Spontaneous Mitotic Recombination in Wild Type and Homologous Recombination Mutants of Candida albicans

Candida albicans, the most common fungal pathogen, is a diploid with a genome that is rich in repeats and has high levels of heterozygosity. To study the role of different recombination pathways on direct-repeat recombination, we replaced either allele of the RAD52 gene (Chr6) with the URA-blaster cassette (hisG-URA3-hisG), measured rates of URA3 loss as resistance to 5-fluoroorotic acid (5FOAR) and used CHEF Southern hybridization and SNP-RFLP analysis to identify recombination mechanisms and their frequency in wildtype and recombination mutants. FOAR rates varied little across different strain backgrounds. In contrast, the type and frequency of mechanisms underlying direct repeat recombination varied greatly. For example, wildtype, rad59 and lig4 strains all displayed a bias for URA3 loss via pop-out/deletion vs. inter-homolog recombination and this bias was reduced in rad51 mutants. In addition, in rad51-derived 5FOAR strains direct repeat recombination was associated with ectopic translocation (5%), chromosome loss/truncation (14%) and inter-homolog recombination (6%). In the absence of RAD52, URA3 loss was mostly due to chromosome loss and truncation (80–90%), and the bias of retained allele frequency points to the presence of a recessive lethal allele on Chr6B. However, a few single-strand annealing (SSA)-like events were identified and these were independent of either Rad59 or Lig4. Finally, the specific sizes of Chr6 truncations suggest that the inserted URA-blaster could represent a fragile site