The NEF4 complex regulates Rad4 levels and utilizes Snf2/Swi2-related ATPase activity for nucleotide excision repair

Nucleotide excision repair factor 4 (NEF4) is required for repair of nontranscribed DNA in Saccharomyces cerevisiae. Rad7 and the Snf2/Swi2-related ATPase Rad16 are NEF4 subunits. We report previously unrecognized similarity between Rad7 and F-box proteins. Rad16 contains a RING domain embedded within its ATPase domain, and the presence of these motifs in NEF4 suggested that NEF4 functions as both an ATPase and an E3 ubiquitin ligase. Mutational analysis provides strong support for this model. The Rad16 ATPase is important for NEF4 function in vivo, and genetic analysis uncovered new interactions between NEF4 and Rad23, a repair factor that links repair to proteasome function. Elc1 is the yeast homologue of a mammalian E3 subunit, and it is a novel component of NEF4. Moreover, the E2s Ubc9 and Ubc13 were linked to the NEF4 repair pathway by genetic criteria. Mutations in NEF4 or Ubc13 result in elevated levels of the DNA damage recognition protein Rad4 and an increase in ubiquitylated species of Rad23. As Rad23 also controls Rad4 levels, these results suggest a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4.     

Genetic analysis of the Neurospora crassa RAD14 homolog mus-43 and the RAD10 homolog mus-44 reveals that they belong to the mus-38 pathway of two nucleotide excision repair systems

Saccharomyces cerevisiae Rad14 and Rad10 proteins are essential for nucleotide excision repair (NER). Rad14 is a UV-damaged DNA binding protein and Rad10 is a structure-specific endonuclease that functions in a complex with Rad1. In this study, we identified and characterized the RAD14 and RAD10 homolog genes in Neurospora crassa, which we named mus-43 and mus-44, respectively. Disruption of mus-43 and mus-44 conferred sensitivity to UV and 4-nitroquinoline 1-oxide, but not to methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, camptothecin, hydroxyurea, or bleomycin. The mus-44 mutant was more sensitive to UV than the mus-43 mutant. Genetic analysis indicated that mus-43 and mus-44 are epistatic to mus-38 which is a homolog of the S. cerevisiae RAD1, but not to mus-18 which belongs to a second excision repair pathway. Immunological assays demonstrated that both mus-43 and mus-44 retained the ability to excise UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts, but that excision ability was completely abolished in the mus-43 mus-18 and mus-44 mus-18 double mutants. These double mutants exhibited extremely high sensitivity to UV. In mus-43 and mus-44 mutants, the UV-induced mutation frequency increased compared to that of the wild-type. The mus-44 mutants also exhibited a partial photoreactivation defect phenotype similar to mus-38. These results suggest that both mus-43 and mus-44 function in the mus-38 NER pathway, but not in the mus-18 excision repair pathway.     

Centrin/Cdc31 is a novel regulator of protein degradation

Rad23 is required for efficient protein degradation and performs an important role in nucleotide excision repair. Saccharomyces cerevisiae Rad23, and its human counterpart (hHR23), are present in a complex containing the DNA repair factor Rad4 (termed XPC, for xeroderma pigmentosum group C, in humans). XPC/hHR23 was also reported to bind centrin-2, a member of the superfamily of calcium-binding EF-hand proteins. We report here that yeast centrin, which is encoded by CDC31, is similarly present in a complex with Rad4/Rad23 (called NEF2). The interaction between Cdc31 and Rad23/Rad4 varied by growth phase and reflected oscillations in Cdc31 levels. Strikingly, a cdc31 mutant that formed a weaker interaction with Rad4 showed sensitivity to UV light. Based on the dual function of Rad23, in both DNA repair and protein degradation, we questioned if Cdc31 also participated in protein degradation. We report here that Cdc31 binds the proteasome and multiubiquitinated proteins through its carboxy-terminal EF-hand motifs. Moreover, cdc31 mutants were highly sensitive to drugs that cause protein damage, failed to efficiently degrade proteolytic substrates, and formed altered interactions with the proteasome. These findings reveal for the first time a new role for centrin/Cdc31 in protein degradation.     

Recognition of DNA damage by XPC coincides with disruption of the XPC-RAD23 complex

The recognition of helix-distorting deoxyribonucleic acid (DNA) lesions by the global genome nucleotide excision repair subpathway is performed by the XPC-RAD23-CEN2 complex. Although it has been established that Rad23 homologs are essential to protect XPC from proteasomal degradation, it is unclear whether RAD23 proteins have a direct role in the recognition of DNA damage. In this paper, we show that the association of XPC with ultraviolet-induced lesions was impaired in the absence of RAD23 proteins. Furthermore, we show that RAD23 proteins rapidly dissociated from XPC upon binding to damaged DNA. Our data suggest that RAD23 proteins facilitate lesion recognition by XPC but do not participate in the downstream DNA repair process.     

Cloning and nucleotide sequence analysis of the Saccharomyces cerevisiae RAD4 gene required for excision repair of UV-damaged DNA

The RAD4 gene of Saccharomyces cerevisiae is required for the incision step of excision repair. We have cloned the RAD4 gene and determined its nucleotide sequence. RAD4 encodes a somewhat basic protein of 754 amino acids (aa) with an Mr of 87,173. RAD4 contains several groups of 4-7 consecutive basic aa residues that could be involved in DNA binding and it also contains an alpha-helix-turn-alpha-helix motif for DNA binding. Like several other DNA repair proteins of S. cerevisiae, the C terminus of RAD4 protein is highly acidic.     

Roles of Rad23 protein in yeast nucleotide excision repair

Nucleotide excision repair (NER) removes many different types of DNA lesions. Most NER proteins are indispensable for repair. In contrast, the yeast Rad23 represents a class of accessory NER proteins, without which NER activity is reduced but not eliminated. In mammals, the complex of HR23B (Rad23 homolog) and XPC (yeast Rad4 homolog) has been suggested to function in the damage recognition step of NER. However, the precise function of Rad23 or HR23B in NER remains unknown. Recently, it was suggested that the primary function of RAD23 protein in NER is its stabilization of XPC protein. Here, we tested the significance of Rad23-mediated Rad4 stabilization in NER, and analyzed the repair and biochemical activities of purified yeast Rad23 protein. Cellular Rad4 was indeed stabilized by Rad23 in the absence of DNA damage. Persistent overexpression of Rad4 in rad23 mutant cells, however, largely failed to complement the ultraviolet sensitivity of the mutant. Consistently, deficient NER in rad23 mutant cell extracts could not be complemented by purified Rad4 protein in vitro. In contrast, partial complementation was observed with purified Rad23 protein. Specific complementation to the level of wild-type repair was achieved by adding purified Rad23 together with small amounts of Rad4 protein to rad23 mutant cell extracts. Purified Rad23 protein was unable to bind to DNA, but stimulated the binding activity of purified Rad4 protein to N-acetyl-2-aminofluorene-damaged DNA. These results support two roles of Rad23 protein in NER: (i) its direct participation in the repair biochemistry, possibly due to its stimulatory activity on Rad4-mediated damage binding/recognition; and (ii) its stabilization of cellular Rad4 protein.     

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis.

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.     

The 26S proteasome negatively regulates the level of overall genomic nucleotide excision repair

Regulation of protein expression can be achieved through destruction of proteins by the 26S proteasome. Cellular processes that are regulated by proteolysis include cell cycle progression, stress responses and differentiation. Several nucleotide excision repair proteins in yeast and humans, such as Rad23, Rad4 and XPB, have been shown to co-purify with Cim3 and Cim5, AAA ATPases of the 19S proteasome regulatory subunit. However, it has not been determined if nucleotide excision repair is regulated through protein destruction. We measured nucleotide excision repair in yeast mutants that are defective in proteasome function and found that the repair of the transcribed and non-transcribed strands of an RNA polymerase II-transcribed reporter gene was increased in the absence of proteasome function. Additionally, overexpression of the Rad4 repair protein, which is bound to the repair/proteolytic factor Rad23, conferred higher rates of nucleotide excision repair. Based on our data we suggest that a protein (or proteins) involved in nucleotide excision repair or in regulation of repair is degraded by the 26S proteasome. We propose that decreased proteasome function enables increased DNA repair, due to the transient accumulation of a specific repair factor, perhaps Rad4.     

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.     

A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

Requirement of Mismatch Repair Genes Msh2 and Msh3 in the Rad1-Rad10 Pathway of Mitotic Recombination in Saccharomyces Cerevisiae

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3Δ mutation has an effect on recombination similar to that of the rad1Δ and rad10Δ mutations. The msh2Δ mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.     

Requirement of RAD52 group genes for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases eta and zeta and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.     

Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.