Chromatin immunoprecipitation with anti-acetyl histone H3 (K9 and K14) and anti-acetyl histone H4 (K5, K8, K12, and K16) antibodies shows that Lys-9 and͞or Lys-14 of histone H3, but not the relevant sites of histone H4 in nucleosomes at the repressed MFA2 promoter, are hyperacetylated after UV irradiation. This level of histone hyperacetylation diminishes gradually as repair proceeds. Accompanying this, chromatin in the promoter becomes more accessible to restriction enzymes after UV irradiation and returns to the pre-UV state gradually. UV-related histone hyperacetylation and chromatin remodeling in the MFA2 promoter depend on Gcn5p and partially on Swi2p, respectively. Deletion of GCN5, but not of SWI2, impairs repair of DNA damage in the MFA2 promoter. The post-UV histone modifications and chromatin remodeling at the repressed MFA2 promoter do not activate MFA2 transcriptionally, nor do they require damage recognition by Rad4p or Rad14p. Furthermore, we show that UV irradiation triggers genome-wide histone hyperacetylation at both histone H3 and H4. These experiments indicate that chromatin at a yeast repressed locus undergoes active change after UV radiation treatment and that failure to achieve histone H3 hyperacetylation impairs the repair of DNA damage.nucleotide ͉ excision repair ͉ Saccharomyces cerevisiae P ackaging DNA into chromatin constrains the genome into the cell nucleus and plays important roles in DNA metabolism. The dynamics of chromatin are finely regulated to control DNA function in response to various stimuli. During activation of gene expression the binding of transcriptional activators and coactivators to promoters results in perturbation of repressive chromatin in the promoter. These events facilitate access of subsequent incoming transcriptional factors to DNA. The changes in repressive chromatin often include chromatin remodeling by various complexes, e.g., SWI͞SNF, and acetylation of histones by histone acetyltransferases, e.g., Gcn5p (1, 2).The regulatory roles of histone acetylation and chromatin remodeling, although well documented, are largely confined to transcription initiation. Their roles in other events such as nucleotide excision repair (NER) that also operates on a chromatin template are only beginning to be explored. NER is a DNA repair pathway with Ͼ30 proteins involved in removing damage from naked DNA (3, 4). Studies have shown that the overall repair of DNA damage by NER is less efficient in reconstituted nucleosomes than in naked DNA (5-7), giving an indication that nucleosomes on damaged DNA inhibit efficient NER. In vivo, early studies with human cells showed that increasing the histone acetylation level overall in chromatin by inhibiting histone deacetylase activities enhances repair synthesis during NER (8-12). Chromatin rearrangement during repair synthesis in NER was also observed as newly repaired DNA in human fibroblasts is more sensitive to nuclease than bulk DNA in chromatin. The nascent repair patch lacks a canonical nucleosome DNase I footprinting, and thi...

The 26S proteasome degrades proteins targeted by the ubiquitin pathway, a function thought to explain its role in cellular processes. The proteasome interacts with the ubiquitin-like N terminus of Rad23, a nucleotide excision repair (NER) protein, in Saccharomyces cerevisiae. Deletion of the ubiquitin-like domain causes UV radiation sensitivity. Here, we show that the ubiquitin-like domain of Rad23 is required for optimal activity of an in vitro NER system. Inhibition of proteasomal ATPases diminishes NER activity in vitro and increases UV sensitivity in vivo. Surprisingly, blockage of protein degradation by the proteasome has no effect on the efficiency of NER. This establishes that the regulatory complex of the proteasome has a function independent of protein degradation.

The Rad23/Rad4 nucleotide excision repair (NER) protein complex functions at an early stage of the NER reaction, possibly promoting the recognition of damaged DNA. Here we show that Rad4 protein is ubiquitinated and degraded in response to ultraviolet (UV) radiation, and identify a novel cullin-based E3 ubiquitin ligase required for this process. We also show that this novel ubiquitin ligase is required for optimal NER. Our results demonstrate that optimal NER correlates with the ubiquitination of Rad4 following UV radiation, but not its subsequent degradation. Furthermore, we show that the ubiquitinproteasome pathway (UPP) regulates NER via two distinct mechanisms. The first occurs independently of de novo protein synthesis, and requires Rad23 and a nonproteolytic function of the 19S regulatory complex of the 26S proteasome. The second requires de novo protein synthesis, and relies on the activity of the newly identified E3 ubiquitin ligase. These studies reveal that, following UV radiation, NER is mediated by nonproteolytic activities of the UPP, via the ubiquitin-like domain of Rad23 and UV radiation-induced ubiquitination of Rad4.

Nucleotide excision repair (NER) promotes the removal of various types of bulky base damage from DNA by a multistage process involving ∼30 different proteins (for review, see Friedberg et al. 1995). The majority of these proteins are highly conserved from yeast to man (for review, see Friedberg et al. 1995). Mutations in NER genes lead to hypersensitivity to killing as well as hypermutability following exposure to DNA-damaging agents such as ultraviolet (UV) radiation. Defective NER in humans predisposes to skin cancer following sunlight exposure, as exemplified by the hereditary NER-defective disease xeroderma pigmentosum (XP). The regulation of NER in living cells is therefore a question of both fundamental and clinical interest. In this study, we demonstrate novel functional relationships between NER and the proteasome in the yeast Saccharomyces cerevisiae.Rad23 protein is one of the multiple proteins involved in NER in S. cerevisiae (Friedberg et al. 1995). The precise function of this protein in this process is not clear.Extracts from cells deleted of the RAD23 gene do not support detectable NER in vitro (Wang et al. 1997;Russell et al. 1999). Such mutants, however, display a level of UV radiation sensitivity that is intermediate between that of wild-type strains and strains deleted for other RAD genes that are indispensable for NER, such as RAD1, RAD2, RAD3, etc. (Watkins et al. 1993;Mueller and Smerdon 1996). In some studies, this intermediate UV radiation sensitivity has been correlated with a partial decrease in NER in vivo (Mueller and Smerdon 1996).Human cells possess two homologs of the yeast RAD23 gene, designated HHR23A and HHR23B (Masutani et al. 1994). HHR23B protein binds tightly to human XPC protein and stimulates the rate of NER in vitro (Masutani et al. 1994(Masutani et al. , 1997Li et al. 1997). On the other hand, deletion of the mouse HHRAD23A or HHRAD23B genes does not result in increased sensitivity to UV radiation in mouse embryo fibroblasts (Friedberg and Meira 2000). Remarkably, mice deleted of the HHRAD23B gene show defective post-natal growth and HHRAD23A HHRAD23B double deletion mutants are inviable (Friedberg and Meira 2000). These observations suggest the existence of as yet unidentified essential functions of HHRAD23 protein, which is partially redundant between the A and B forms.Levels of human HHRAD23A protein are regulated in