Project description:DNA damage response (DDR) plays pivotal roles in maintaining genome integrity and stability. An effective DDR requires the involvement of hundreds of genes that compose a complicated network. To identify novel genes involved in DDR, we screened a genome-wide Schizosaccharomyces pombe (S. pombe) haploid deletion library against six different DNA damage reagents. We identified 52 genes that were actively involved in DDR. Among the 52 genes, 20 genes were linked to DDR for the first time. For a better understanding of DDR genes function, we performed a DNA microarray assay to analyze the gene expression profiles of eight deletions.
Project description:Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. Our project is to show that the conserved RNA-binding Seb1 is a pausing-promoting factor for RNA polymerases I to control cotranscriptional RNA processing. Here, we did a proximity dependent biotinylation followed by mass spectrometry (PDB-MS) of the Seb1 protein in order to assess for physical interactions with the RNAPI transcription machinery. A mutant E. coli BirA enzyme is fused to the Seb1 protein. This mutant version of BirA uses biotin to catalyze the formation of biotinoyl-5′-AMP (bioAMP), thereby generating a ‘cloud’ of activated biotin molecules that can react with free primary amines (most often lysine residues) of neighboring proteins. This experiment will support the conclusion that Seb1 is located at the rDNA locus.
