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

Chromatin immunoprecipitation (ChIP) assay in Candida albicans

Chromatin immunoprecipitation (ChIP) is a widely used technique which can determine the in vivo association of a specific protein on a particular DNA locus in the genome. In this method cross-linked chromatin is sheared and immunoprecipitated with antibodies raised against a target protein of interest. The end result of this process is the enrichment of DNA fragments associated with the desired protein. Thus, interactions between proteins and genomic loci in cellular context can be determined by this technique. Here, we are describing a ChIP protocol that is optimized for Candida albicans. The protocol requires 4–5 days for completion of the assay and has been used to produce robust ChIP results for diverse proteins in this organism and its related species including Candida dubliniensis and Candida tropicalis

Replication forks stall/terminate randomly during centromere replication.

<p>Schematic of a ∼30 kb region of chromosome 7 centered on the centromere (<i>CEN7</i>) is shown. The hatched rectangles denote the positions of the nearest neocentromere (<i>nCEN7</i>) hotspots as described earlier <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Thakur1" target="_blank">[38]</a>. The filled grey circles indicate the positions of the chromosomal origins identified during 2D analysis (<i>ORI7-LI</i> and <i>ORI7-RI</i>). Replication intermediates from this 30 kb region in asynchronously grown <i>C. albicans</i> cells were analyzed by 2D gel electrophoresis assays using overlapping restriction fragments (1–8). Arrowheads and numbers indicate the positions and the identities of the ORFs. Open rectangles indicate the fragments used for the <i>ARS</i> function assay. Schematic of replication intermediates indicates simple ‘Y’ arcs (broken line), specific termination (Double-Ys), joint molecules (Xs) and random termination signals (triangular smear). The dark grey zone at the inflection point of the ‘Y’ arc indicates replication fork stalling. The presence of Xs and triangular smear in fragments 1, 2, 4, 5 and 7 indicates replication fork stalling/termination. Bubble arcs are observed in fragments 3 and 6 signaling chromosomal origins of replication (<i>ORI7-LI</i> and <i>ORI7-RI</i>). The plate pictures in the lower panel show the results of an <i>ARS</i> function assay using the fragments (open rectangles) located within <i>ORI7-LI</i> and <i>ORI7-RI</i> in the wild-type. The corresponding 2-D signals in high contrast are shown in the inset. Both fragments show <i>ARS</i> activity.</p

Rad51 or Rad52 depletion affects kinetochore assembly.

<p>(A) Percentage of cells at each cell cycle stage was determined for wild-type, <i>rad51</i> and <i>rad52</i> mutants. At least 100 cells were counted at each stage. lb, large bud; elb, extended large bud. The extended large bud (elb) is an aberrant G2/M phenotype observed in <i>rad51</i> and <i>rad52</i> mutants only. (B) Using confocal microscopy, GFP-CENP-A^CaCse4 foci were scored in large budded cells of wild-type, <i>rad51</i> or <i>rad52</i> mutant strains. They were classified into three categories as shown in figure. n≥100. The percentage of large budded cells under each category was calculated for wild-type and mutant strains, and plotted. An increase in percentage of large budded cells with declustered GFP-CENP-A^CaCse4signals is observed in <i>rad51</i> or <i>rad52</i> mutant, which is an indicator of improper kinetochore assembly <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Thakur2" target="_blank">[44]</a>. Bar (white line), 5 µm. (C) Intensity of the GFP-CENP-A^CaCse4 spots was measured by the Image J software for wild-type, <i>rad51</i> or <i>rad52</i> mutant cells for the G2/M stage, n = 10 in each case. The normalized mean GFP intensity (with respect to background) was calculated for each cell and plotted. Legend shows the different categories of strains and stages and the average GFP-CENP-A^CaCse4 intensity ± S.E.M. Associated DIC images show the measurement technique for calculating GFP intensity. Bar (white line), 1 µm.</p

A replication-coupled repair based model of centromere inheritance.

<p>(A) A replication-coupled repair based model for propagation of CENP-A^CaCse4 chromatin at an early replicating centromere. During S phase, replication forks, originating from proximal conserved early origins, stall at the kinetochore. The stalling of replication forks at the centromere leads to accumulation of single stranded (ss) DNA. The homologous recombination proteins Rad51 and Rad52 are possibly recruited via ssDNA to the stalled replication forks at the centromere. A transient Rad51/Rad52-CENP-A^CaCse4 complex is stabilized by one or more cell-cycle regulated proteins (chaperone?) at the centromere, thereby regulating the replication coupled deposition of CENP-A at the centromeres. The CENP-A^CaCse4 bound kinetochore is indicated by red circles whereas replisome is depicted by large purple circle. Functional origin locations are shown by blue filled ovals. <i>CEN</i>- centromere, KT- kinetochore. (B) Schematic depicts the effect of CENP-A^CaCse4/Rad51/Rad52 depletion on replication fork passage through the centromere. Depletion of CENP-A^CaCse4 causes kinetochore disintegrity. As a result forks are no longer stalled at the <i>CEN</i>-kinetochore barrier and fork stalling is weakened. Depletion of Rad51/Rad52 also causes improper kinetochore assembly. As a result fork stalling is weakened. (C) Schematic depicts the effect of deletion of a proximal origin on replication fork passage through the centromere. On deletion of a proximal origin, fork stalling at the centromere is reduced and concomitantly CENP-A^CaCse4 binding is reduced, leading to a weaker centromere.</p

Centromeric fork stalling/termination is CENP-A mediated and involves Rad51 and Rad52.

<p>(A) A line diagram of a 6 kb region of chromosome 7 centered on <i>CEN7</i> is shown. <i>CEN7</i> (black rectangle) and flanking regions (grey rectangles) that include the 5 kb <i>Eco</i>RI fragment (fragment 4 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a>) used for 2D gel analysis are shown. Schematics of replication intermediates as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a> are also shown. Quantification of the termination signals was performed as following: Relative intensity of termination (RIT) = random termination signal/1n spot. (B) Replication intermediates from the core <i>CEN7</i> region were determined by 2-D gel analysis at wild-type and depleted levels of CENP-A^CaCse4. (C) The 1n spot (schematic) and the termination signals (triangular smear) were quantified by Image Gauge software (Fujifilm) and RIT values were calculated as described above for wild-type and CENP-A^CaCse4 depleted condition. The RIT values, plotted on a bar graph, indicate a gradual decrease in the termination signal in CENP-A^CaCse4 repressed conditions as compared to wild-type. The values represent the mean of three independent 2D experiments ± SD. (D) Replication intermediates from the core <i>CEN7</i> region (black rectangle) were determined by 2D gel analysis for the wild-type, <i>rad51</i>, and <i>rad52</i> mutants. (E) RIT values were calculated for wild-type and <i>rad51</i> and <i>rad52</i> mutants. The RIT values, plotted on a bar graph, indicate a decrease in the termination signal in <i>rad51</i> and <i>rad52</i> mutants as compared to wild-type. The values represent the mean of three independent 2D experiments ± SD.</p

Replication-segregation interaction is evolutionarily conserved in unicellular organisms.

<p>(A) The phylogenetic tree reflects the evolutionary relationships of the corresponding taxa. The tree is drawn to scale with branch lengths in the units of the number of base substitutions per site in the 23S or 25S rRNA nucleotide sequences of the four species. (B) <i>CEN</i>-like loci or <i>CEN</i>s (green boxes) in prokaryotes and unicellular eukaryotes respectively are flanked by early replication origins (pink circles). The blue circles indicate the centromere factors influencing origin activity. The yellow circles indicate the origin/replication associated factors influencing <i>CEN</i> function. In the genome of the bacteria <i>B. subtilis</i>, the single replication origin is flanked by <i>CEN</i>-like <i>parS</i> sequence. The Spo0J (ParB) protein, binding to <i>parS</i>, organizes <i>ori</i> activity as well as recruits Smc proteins for proper segregation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Gruber1" target="_blank">[17]</a>. In <i>S. cerevisiae</i>, which has short ‘point’ centromeres, the Ctf19 complex directly recruits initiation factors for early firing of proximal origins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Natsume1" target="_blank">[20]</a>. Although early firing has been suggested for playing a role in <i>CEN</i> function, no <i>cis</i> factors has been identified. In <i>C. albicans</i>, which has ‘short regional’ <i>CEN</i>s, <i>CEN</i>s have been shown to govern early replication of proximal origins, although no <i>cis</i> factors were identified <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Koren1" target="_blank">[5]</a>. In this study we show that fork stalling at <i>CENs</i> from proximal origins recruit Rad51/Rad52 that, in turn, regulates CENP-A deposition. Finally in the ‘large regional’ centromeres of <i>S. pombe</i>, the centromeric heterochromatic protein Swi6 activates pericentric replication origins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Hayashi1" target="_blank">[16]</a>. The fork protection complex (FPC) that travels with the replisome negatively regulates Ams2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Takayama2" target="_blank">[73]</a> that, in turn, regulates CENP-A deposition <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Takayama1" target="_blank">[10]</a>.</p

Rad51 and Rad52 aid in CENP-A^CaCse4 recruitment.

<p>(A) Standard ChIP assays followed by quantitative real time PCR (qPCR) were performed in wild-type, <i>rad51</i> or <i>rad52</i> for CENP-A^CaCse4-Prot A for <i>CEN5</i> and <i>CEN7</i>. qPCR amplification from a non-centromeric (<i>non-CEN</i>) control was also performed to detect the background DNA elution in the ChIP assays. Enrichment of CENP-A^CaCse4 at the centromeres was calculated as a percentage of the total chromatin input and values were plotted as mean of three independent ChIP experiments ± SD. (B) Western blot analysis performed with the whole cell lysates from wild-type and homologous recombination (HR) and non-homologous end joining (NHEJ) mutants using anti-CENP-A^CaCse4 antibodies. PSTAIRE was used as a loading control. The relative levels of CENP-A^CaCse4 (CENP-A^CaCse4/PSTAIRE) was computed for each mutant and plotted in a bar graph. (C) Co-immunoprecipitation assays for the two sets of strains carrying Rad51-V5 and Rad52-V5 were performed using anti-V5 antibodies. Precipitates were analyzed by western blotting with anti- CENP-A^CaCse4 antibodies. In each case, untagged strains and no (-) antibody fractions were used as controls. Blue asterisk indicates a non-specific band.</p

Centromere proximal origins maintain centromere functioning.

<p>(A) Schematic showing the strategy of deletion of <i>ORI7-RI</i> (shown as red circles) and the chromosome loss assay. Since the strain RM100AH <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Sanyal1" target="_blank">[33]</a> is heterozygous for both <i>HIS1</i> and <i>ARG4</i> that mark the two chromosome 7 homologs, replacement of <i>ORI7-RI</i> by <i>URA3</i> will create a strain that can be used to assay the loss of the altered chromosome (by scoring simultaneous loss of Ura and Arg or Ura and His markers). (B) Dilutions of <i>ORI7-RI</i> deleted transformants (<i>ORI7-RI/</i>Δ<i>ORI7-RI</i>) were spotted on CM+5′-FOA plates to estimate the chromosome loss frequency. USN148 (Δ<i>ura3::imm434/</i>Δ<i>ura3::imm434</i>/CIp10) strain was used as the wild-type control to estimate the spontaneous loss rate of a chromosome. Subsequently, the 5′-FOA positive colonies were patched on YPDU. From YPDU these colonies were re-patched onto CM- Ura, CM-His and CM-Arg plates to assay for the loss of the altered chromosome 7 homolog. (C) Standard ChIP assays followed by quantitative real time PCR (qPCR) were performed in wild-type and CAKS105 (Δ<i>ORI7-RI/</i>Δ<i>ORI7-RI</i>) strain for enrichment of CENP-A^CaCse4-Prot A at the core <i>CEN7</i>. Enrichment of CENP-A^CaCse4 at the centromere was calculated as a percentage of the total chromatin input and values were plotted as mean of two independent experiments (three technical replicates for each experiment) ± SD. (D) Line diagrams depicting ∼15 kb region surrounding <i>CEN7</i> are shown (symbols as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a>). Schematics depict the replication intermediates as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a>. Bar (black line), 1 kb. The upper panel shows the 2-D image from the core <i>CEN7</i> region (fragment 4 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a>) in the wild-type. The lower panel shows the 2-D image from the same fragment when <i>ORI7-RI</i> is deleted in CAKS105 (Δ<i>ORI7-RI/</i>Δ<i>ORI7-RI</i>).</p