Project description:The stability of the genome is occasionally challenged by the formation of DNA-RNA hybrids and R-loops, which can be influenced by the chromatin context. This is mainly due to the fact that DNA-RNA hybrids hamper the progression of replication forks, leading to fork stalling and, ultimately, DNA breaks. Through a specific screening of chromatin modifiers performed in the yeast Saccharomyces cerevisiae, we have found that the Rtt109 histone acetyltransferase is involved in several steps of R-loop-metabolism and their associated genetic instability. On one hand, Rtt109 prevents DNA-RNA hybridization by the acetylation of histone H3 lysines 14 and 23, and on the other hand, it is involved in the repair of replication-born DNA breaks, such as those that can be caused by R-loops, by acetylating lysines 14 and 56. In addition, Rtt109 loss renders cells highly sensitive to replication stress in combination with R-loop-accumulating THO-complex mutants. Our data evidence that the chromatin context simultaneously influences the occurrence of DNA-RNA hybrid-associated DNA damage and its repair, adding complexity to the source of R-loop-associated genetic instability.
Project description:The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we report a microarray expression study of the protein kinase Pkc1, histone acetyl transferase Rtt109 and histone H3 in Saccharomyces cerevisiae under conditions of replicative stress.
Project description:RPA12 is a subunit of RNA polymerase I. We used microarrays to know the effect RPA12 deltion in lipid metabolism and identified distinct classes of up-regulated genes during this process.
Project description:Spn1/Iws1 is an essential eukaryotic transcription elongation factor that is conserved from yeast to humans. Several studies have shown that Spn1 functions as a histone chaperone to control transcription, RNA splicing, genome stability, and histone modifications as an integral member of the RNA polymerase II elongation complex. However, the precise role of Spn1 is not understood, and there is little understanding of why it is essential for viability. To address these issues, we have isolated eight suppressor mutations that bypass the essential requirement for Spn1 in Saccharomyces cerevisiae. Unexpectedly, the suppressors identify several functionally distinct complexes and activities, including the histone chaperone FACT, the histone methyltransferase Set2, the Rpd3S histone deacetylase complex, the histone acetyltransferase Rtt109, the nucleosome remodeler Chd1, and a member of the SAGA co-activator complex, Sgf73. The identification of these distinct groups and their analysis suggests that there are multiple mechanisms by which Spn1 bypass can occur, including changes in histone acetylation and alterations of other histone chaperones. Thus, Spn1 may participate in multiple functions during transcription. Our results suggest that bypass of a subset of these functions allows viability in the absence of Spn1.
Project description:Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rtt109 for C. albicans pathogenicity, we deleted the predicted homologue of Rtt109 in the clinical C. albicans isolate, SC5314. C. albicans rtt109 -/- mutant cells lack acetylated H3K56 (H3K56ac) and are hypersensitive to genotoxic agents. Additionally, rtt109 -/- mutant cells constitutively display increased H2A S129 phosphorylation and elevated DNA repair gene expression, consistent with endogenous DNA damage.
Project description:RPA12 is a subunit of RNA polymerase I. We used microarrays to know the effect RPA12 deltion in lipid metabolism and identified distinct classes of up-regulated genes during this process. Stationary phase cells were taken for RNA extraction and hybridization on Affymetric microarrays