Project description:The Rad23/Rad4 protein complex plays a major role in DNA damage recognition during nucleotide excision repair (NER) in yeast. We recently showed that two distinct pathways contribute to efficient NER in yeast. The first operates independently of de novo protein synthesis and requires a nonproteolytic function of the 19S regulatory complex of the 26S proteasome and Rad23. The second pathway requires de novo protein synthesis, and relies on the activity of a newly identified Rad7-containing E3 ubiquitin ligase that ubiquitinates Rad4 in response to UV. Surprisingly, we found that cells deleted of either Rad23 or Rad4 caused reduced Rad4 and Rad23 mRNA levels respectively. We considered the possibility of an unexpected role of Rad23 and Rad4 in regulating the expression of genes involved in the transcriptional response to DNA damage. Gene expression profiling has suggested that Rad23 and Rad4 may function as a complex to affect transcription of a small subset of genes in response to UV damage. To determine how Rad4 and Rad23 contribute to the regulation of these genes, we have examined the occupancy of Rad4/Rad23 in their promoter regions by chromatin immunoprecipitation (ChIP), both in the presence and absence of UV damage. Our preliminary ChIP data suggests that the Rad4/Rad23 complex regulates a set of genes in response to UV light. We also proposed that the transcriptional regulatory activity of the Rad4-Rad23 complex required Rad4 ubiquitination. These arrays test this theory using the psocs mutant strain, which is unable to facilitate Rad4 ubiquitination after UV irradiation. *** This Series represents the gene expression component of the study. Expression analysis was performed on the pRAD7 strain, which served as the WT control, and harboured the WT RAD7 sequence on the pRS313 plasmid, the psocs strain, which harboured the same plasmid with 2 point mutations in the RAD7 sequence that prevented post-UV Rad4 ubiquitination. Expression analysis was also conducted on a pRAD7 and psocs Strain with RAD23 deleted. Analysis was performed using untreated strains, and strains 15 minutes and 60 minutes after 100Jm-2 UV irradiation. mRNA was extracted from logaritmically growing cells.

Project description:Transcription-coupled nucleotide excision repair (TC-NER) is promptly triggered by 'local' elongating RNAP II molecules encountering DNA adducts such as cyclobutane pyrimidine dimers (CPD) induced by ultraviolet light and speeds up removing and repair in transcribed loci. Genome-wide measurements have revealed a dramatic shutdown of global transcription with a few exceptions of individual genes that are consequently highly upregulated by DNA damage. Deciphering the underlying mechanisms of the multifaceted transcriptional response always comprises one of the most fascinating frontiers in DNA repair research. Recent reports have revealed that persistent RNAP II initiation at the transcription start site of active regulatory regions with the continuous synthesis of start-RNAs guarantee sufficient scanning and repair of the whole transcribed genome, although transcription elongation drastically slows down shortly after genotoxic stress. Furthermore, a recent study found that RNA is extremely rapidly m6A methylated by METTL3 in response to UV irradiation which is crucial for recruitment of Pol k to DNA damage sites and subsequently mediates TC-NER. Together, these findings substantiate the molecular processes underlying the transcription-coordinated cellular response upon genotoxic stress might be more complex than previously believed. Unfortunately, all these findings merely focused on the early phase of recovery (within 4 hr after DNA damage). It remains to be determined how accessible chromatin reconfigures and regulates transcriptional programs in UV-induced DNA damage repair. It is also unclear whether the dynamics of gene expression are associated with other epigenomic reconstruction events such as global DNA methylation and demethylation. Moreover, the roles of internal messenger RNA modifications like N6-methyladenosine in TC-NER are poorly understood. Our integrative analyses provide a comprehensive view of the spatio-temporal chromatin configuration and epigenetic characterizations that accompanies TC-NER.

Dataset Information

Transcription profiling of Saccharomyces cerevisiae to investigate the role of rad23/rad4 protein complex in transcription and DNA repair in yeast

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