RNA Polymerase I Transcription Factors in Active Yeast rRNA Gene Promoters Enhance UV Damage Formation and Inhibit Repair

Abstract

UV photofootprinting and repair of pyrimidine dimers by photolyase was used to investigate chromatin structure, protein-DNA interactions, and DNA repair in the spacer and promoter of Saccharomyces cerevisiae rRNA genes. Saccharomyces cerevisiae contains about 150 copies of rRNA genes separated by nontranscribed spacers. Under exponential growth conditions about half of the genes are transcribed by RNA polymerase I (RNAP-I). Initiation of transcription requires the assembly of the upstream activating factor (UAF), the core factor (CF), TATA binding protein, and RNAP-I with Rrn3p on the upstream element and core promoter. We show that UV irradiation of wild-type cells and transcription factor mutants generates photofootprints in the promoter elements. The core footprint depends on UAF, while the UAF footprint was also detected in absence of the CFs. Fractionation of active and inactive promoters showed the core footprint mainly in the active fraction and similar UAF footprints in both fractions. DNA repair by photolyase was strongly inhibited in active promoters but efficient in inactive promoters. The data suggest that UAF is present in vivo in active and inactive promoters and that recruitment of CF and RNAP-I to active promoters generates a stable complex which inhibits repair.In Saccharomyces cerevisiae, about 150 copies of rRNA genes are clustered as tandem repeats in the nucleolus. Each repeat contains a gene for the 35S rRNA transcribed by RNA polymerase I (RNAP-I) and a gene for the 5S rRNA transcribed by RNA polymerase III. A nontranscribed spacer 1 (NTS1) extends from the transcription termination site to the 5S gene and includes the enhancer. A nontranscribed spacer 2 (NTS2) spans the region between the 5S gene and the start of the 35S gene and contains a ribosomal origin of replication (rARS) and the promoter with the core and the upstream elements ( Fig. 1) (35, 36).rRNA genes are transcribed with high efficiency to keep up with the cell's metabolic activity and demand for ribosomes (19). The rate of rRNA synthesis is regulated as a response to the cellular environment, either by a change of the initiation rate in transcriptionally competent genes (3, 15) or by activation and inactivation of additional gene copies (11,41). The transcribed genes are free of nucleosomes ("open"), while the inactive genes are packaged in nucleosomes ("closed"). The spacer region is always nucleosomal (11). Nucleosomes are positioned in NTS2 between the promoter and the 5S gene (18,28,52). A nucleosome with multiple positions was reported previously for the 5S gene (7).Transcription initiation involves coordinated interactions of at least four transcription factors with promoter elements and RNAP-I: the upstream activating factor (UAF) containing Rrn5, Rrn9, Rrn10, the H3 and H4 histones and Uaf30p; the core factor (CF) containing Rrn6, Rrn7, and Rrn11; TBP, the TATA binding protein; and Rrn3p, a factor that binds RNAP-I (reviewed in references 35 and 36). TFIIH and CSB are additional factors that are involved in RNAP-I t...

Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin

“…1A, lower panel [39]). Like NER (see description above), photolyase repair of CPDs in the yeast rDNA sequences is hindered by the presence of nucleosomes, and photoreactivation experiments confirmed the presence of CF and UAF at rDNA regulatory regions (32,33). In WT cells, NER is slow over the entire RNAPI promoter and downstream of the transcription initiation site up to nucleotide ϩ34 (Fig.…”

UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes

“…However, whereas UAF binds to promoters of both inactive and active rRNA genes, CF mostly binds to the promoters of active rRNA genes (25,26). To define the presence of RNAPI pre-initiation complex after UV irradiation, the association of HA tagged UAF and CF with the rDNA promoter of WT ( NER + ) cells was analyzed by ChIP.…”

Transcription of Multiple Yeast Ribosomal DNA Genes Requires Targeting of UAF to the Promoter by Uaf30

“…However, it is not known if they are modified in the context of UAF. Since UAF is bound to the rDNA promoter of both active and inactive repeats in WT cells (24) and even remains associated with the rDNA promoter during stationary phase (8), posttranslational modification of H3, H4, or one of the other UAF subunits could be a potential mechanism for controlling the activation of genes in response to altered nutrient levels through modulating UAF function (18,34). Analogously, the negative impact that deleting UAF30 has on UAF function results in a decrease in the number of active rDNA genes.…”