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UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

ABSTRACT: Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage.

SUBMITTER: Zhou Z

PROVIDER: S-EPMC4551923 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

Zhou Zheng Z Humphryes Neil N van Eijk Patrick P Waters Raymond R Yu Shirong S Kraehenbuehl Rolf R Hartsuiker Edgar E Reed Simon H SH

Nucleic acids research 20150706 15

Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes includ ...[more]

PMID: 26150418

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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:In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicansIMPORTANCECandida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

Dataset Information

UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

Publications

UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

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