Project description:Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage-bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage-bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domains-containing Saccharomyces cerevisiae protein, Esc2, facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause sub-optimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress, while globally prevented at undamaged replicating chromosomes.
Project description:In the yeast Saccharomyces cerevisiae, cleavage factor I (CFI) and cleavage and polyadenylation factor (CPF) build the core of the transcription termination machinery. CFI comprises the Rna14, Rna15, Pcf11, and Clp1 proteins, as well as the associated Hrp5 RNA-binding protein. We found that CFI participates in the DNA damage response and that rna14-1 shows synthetic growth defects with mutants of different repair pathways, including homologous recombination, non-homologous end joining, post replicative repair, mismatch repair, and nucleotide excision repair, implicating that impaired RNAPII termination and 3â-end processing decreases the cellular tolerance for DNA damage. Beyond replication progression defects, we found that bypass of the G1/S checkpoint in rna14-1 cells leads to synthetic sickness, accumulation of phosphorylated H2A, as well as increase in Rad52-foci and in recombination. Our data provide evidence that CFI dysfunction impairs RNAPII turnover, leading to replication hindrance and lower tolerance to exogenous DNA damage. These findings underscore the importance of coordination between transcription termination, DNA repair and replication in the maintenance of genomic stability. S. cerevisiae strains were grown in YPAD liquid culture at 30°C, total RNA was isolated and hybridized on Affymetrix microarrays.
Project description:We expressed human RECQL5 in the yeast model Sacharomyces cerevisiae. Being RECQL5 the only member of the family shown to interact with the RNA polymerase II we explored the recruitment of RECQL5 to chromatin. Data reveal a genome wide recruitment of RECQL5 at actively transcribed regions that increases toward the 3’ end of the gene.
Project description:In the yeast Saccharomyces cerevisiae, cleavage factor I (CFI) and cleavage and polyadenylation factor (CPF) build the core of the transcription termination machinery. CFI comprises the Rna14, Rna15, Pcf11, and Clp1 proteins, as well as the associated Hrp5 RNA-binding protein. We found that CFI participates in the DNA damage response and that rna14-1 shows synthetic growth defects with mutants of different repair pathways, including homologous recombination, non-homologous end joining, post replicative repair, mismatch repair, and nucleotide excision repair, implicating that impaired RNAPII termination and 3’-end processing decreases the cellular tolerance for DNA damage. Beyond replication progression defects, we found that bypass of the G1/S checkpoint in rna14-1 cells leads to synthetic sickness, accumulation of phosphorylated H2A, as well as increase in Rad52-foci and in recombination. Our data provide evidence that CFI dysfunction impairs RNAPII turnover, leading to replication hindrance and lower tolerance to exogenous DNA damage. These findings underscore the importance of coordination between transcription termination, DNA repair and replication in the maintenance of genomic stability.
Project description:Transcriptional profiling of Saccharomyces cerevisiae cells comparing the W303-1A wildtype with the W303-1A double mutant for MSN2 and MSN4 during zinc deficient conditions Keywords: Genetic modification with zinc limitation
Project description:The yeast Saccharomyces cerevisiae has emerged as a superior model organism. Selection of distinct laboratory strains of S. cerevisiae with unique phenotypic properties, such as superior mating or sporulation efficiencies, has facilitated advancements in research. W303 is one such laboratory strain that is closely related to the first completely sequenced yeast strain, S288C. In this work, we provide a high-quality, annotated genome sequence for W303 for utilization in comparative analyses and genome-wide studies. Approximately 9500 variations exist between S288C and W303, affecting the protein sequences of ∼700 genes. A listing of the polymorphisms and divergent genes is provided for researchers interested in identifying the genetic basis for phenotypic differences between W303 and S288C. Several divergent functional gene families were identified, including flocculation and sporulation genes, likely representing selection for desirable laboratory phenotypes. Interestingly, remnants of ancestor wine strains were found on several chromosomes. Finally, as a test of the utility of the high-quality reference genome, variant mapping revealed more accurate identification of accumulated mutations in passaged mismatch repair-defective strains.
Project description:Saccharomyces cerevisiae strain W303 is a widely used model organism. However, little is known about its genetic origins, as it was created in the 1970s from crossing yeast strains of uncertain genealogy. To obtain insights into its ancestry and physiology, we sequenced the genome of its variant W303-K6001, a yeast model of ageing research. The combination of two next-generation sequencing (NGS) technologies (Illumina and Roche/454 sequencing) yielded an 11.8 Mb genome assembly at an N50 contig length of 262 kb. Although sequencing was substantially more precise and sensitive than whole-genome tiling arrays, both NGS platforms produced a number of false positives. At a 378× average coverage, only 74 per cent of called differences to the S288c reference genome were confirmed by both techniques. The consensus W303-K6001 genome differs in 8133 positions from S288c, predicting altered amino acid sequence in 799 proteins, including factors of ageing and stress resistance. The W303-K6001 (85.4%) genome is virtually identical (less than equal to 0.5 variations per kb) to S288c, and thus originates in the same ancestor. Non-S288c regions distribute unequally over the genome, with chromosome XVI the most (99.6%) and chromosome XI the least (54.5%) S288c-like. Several of these clusters are shared with ?1278B, another widely used S288c-related model, indicating that these strains share a second ancestor. Thus, the W303-K6001 genome pictures details of complex genetic relationships between the model strains that date back to the early days of experimental yeast genetics. Moreover, this study underlines the necessity of combining multiple NGS and genome-assembling techniques for achieving accurate variant calling in genomic studies.
Project description:These three replicates were analyzed in "Genomewide identification of Sko1 target promoters reveals a regulatory network that operates in response to osmotic stress in Saccharomyces cerevisiae. ", by Proft M, Gibbons FD, Copeland M, Roth FP, Struhl K; published in Eukaryot Cell. 2005 Aug;4(8):1343-52. A new analysis algorithm for Chip-chip data ('Chipper') is described in Genome Biology. Manuscript entitled "Chipper: discovering transcription-factor targets from chromatin immunoprecipitation microarrays using variance stabilization." by FD Gibbons, M Proft, K Struhl, and FP Roth. Accepted, no publication date as yet. Keywords: ChIP-chip
Project description:Transcriptional profiling of Saccharomyces cerevisiae cells comparing the W303-1A wildtype with the W303-1A double mutant for MSN2 and MSN4 during zinc deficient conditions Keywords: Genetic modification with zinc limitation Two condition experiment, W303-1A vs W303-1A delta MSN2, MSN4. Biological replicates: 2 wildtype, 2 knock-out, independently grown and harvested.