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1.
Xie Z  Liu S  Zhang Y  Wang Z 《Nucleic acids research》2004,32(20):5981-5990
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.  相似文献   

2.
A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.  相似文献   

3.
A protein that exemplifies the intimate link between the ubiquitin/proteasome system (UPS) and DNA repair is the yeast nucleotide excision repair (NER) protein Rad23 and its human orthologs hHR23A and hHR23B. Rad23, which was originally identified as an important factor involved in the recognition of DNA lesions, also plays a central role in targeting ubiquitylated proteins for proteasomal degradation, an activity that it shares with other ubiquitin receptors like Dsk2 and Ddi1. Although the finding that Rad23 serves as a ubiquitin receptor explains to a large extent its importance in proteasomal degradation, the precise mode of action of Rad23 in NER and the possible link with the UPS is less clear. In this review, we discuss our present knowledge on the functions of Rad23 in protein degradation and DNA repair and speculate on the importance of the dual roles of Rad23 for the cell's ability to cope with stress conditions.  相似文献   

4.
The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.  相似文献   

5.
A role for Rad23 proteins in 26S proteasome-dependent protein degradation?   总被引:4,自引:0,他引:4  
Treatment of cells with genotoxic agents affects protein degradation in both positive and negative ways. Exposure of S. cerevisiae to the alkylating agent MMS resulted in activation of genes that are involved in ubiquitin- and 26S proteasome-dependent protein degradation. This process partially overlaps with the activation of the ER-associated protein degradation pathway. The DNA repair protein Rad23p and its mammalian homologues have been shown to inhibit degradation of specific substrates in response to DNA damage. Particularly the recently identified inhibition of degradation by mouse Rad23 protein (mHR23) of the associated nucleotide excision repair protein XPC was shown to stimulate DNA repair.Recently, it was shown that Rad23p and the mouse homologue mHR23B also associate with Png1p, a deglycosylation enzyme. Png1p-mediated deglycosylation plays a role in ER-associated protein degradation after accumulation of malfolded proteins in the endoplasmic reticulum. Thus, if stabilization of proteins that are associated with the C-terminus of Rad23p is a general phenomenon, then Rad23 might be implicated in the stimulation of ER-associated protein degradation as well. Interestingly, the recently identified HHR23-like protein Mif1 is also thought to play a role in ER-associated protein degradation. The MIF1 gene is strongly activated in response to ER-stress. Mif1 contains a ubiquitin-like domain which is most probably involved in binding to S5a, a subunit of the 19S regulatory complex of the 26S proteasome. On the basis of its localization in the ER-membrane, it is hypothesized that Mif1 could play a role in the translocation of the 26S proteasome towards the ER-membrane, thereby enhancing ER-associated protein degradation.  相似文献   

6.
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.  相似文献   

7.
Following DNA damage cells initiate cell cycle checkpoints to allow time to repair sustained lesions. Rad9, Rad1, and Hus1 proteins form a toroidal complex, termed the 9-1-1 complex, that is involved in checkpoint signaling. 9-1-1 shares high structural similarity to the DNA replication protein proliferating cell nuclear antigen (PCNA) and 9-1-1 has been shown in vitro to stimulate steps of the repair process known as long patch base excision repair. Using a system that allows conditional repression of the Rad9 protein in human cell culture, we show that Rad9, and by extension, the 9-1-1 complex, enhances cell survival, is required for efficient exit from G2-phase arrest, and stimulates the repair of damaged DNA following ionizing radiation. These data provide in vivo evidence that the human 9-1-1 complex participates in DNA repair in addition to its previously described role in DNA damage sensing.  相似文献   

8.
Schärer OD 《Molecular cell》2007,28(2):184-186
The structure of the Rad4/Rad23 protein, shown in a recent issue of Nature (Min and Pavletich, 2007), reveals how structurally diverse lesions are recognized in eukaryotic nucleotide excision repair: by probing for accessible nondamaged DNA opposite the lesion.  相似文献   

9.
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.  相似文献   

10.
Human 3-methyladenine-DNA glycosylase (MPG protein) initiates base excision repair by severing the glycosylic bond of numerous damaged bases. In comparison, homologues of the Rad23 proteins (hHR23) and the hXPC protein are involved in the recognition of damaged bases in global genome repair, a subset of nucleotide excision repair. In this report, we show that the hHR23A and -B also interact with the MPG protein and can serve as accessory proteins for DNA damage recognition in base excision repair. Furthermore, the MPG.hHR23 protein complex elevates the rate of MPG protein-catalyzed excision from hypoxanthine-containing substrates. This increased excision rate is correlated with a greater binding affinity of the MPG protein-hHR23 protein complex for damaged DNA. These data suggest that the hHR23 proteins function as universal DNA damage recognition accessory proteins in both of these major excision repair pathways.  相似文献   

11.
Aims: Lesions of DNA are removed by nucleotide excision repair (NER) process in the living systems. NER process‐related host factors are believed to aid recovery steps during viral integration. Here, we report identification and characterization of a DNA repair molecule Rad23 from kuruma shrimp Marsupenaeus japonicus. Methods and Results: The full‐length cDNA of M. japonicus Rad23 gene (MjRad23) has 1149 bp coding for a putative protein of 382 amino acids with a 5′ untranslated region (UTR) of 92 bp and 3′ UTR region of 1116 bp. Quantitative expression analysis revealed MjRad23 is constitutively expressed in all the organs of healthy shrimp, whereas with high level in muscle tissue. Although MjRad23 expression is observed in every haemolymph samplings to post‐white spot syndrome virus infection, high expression is recorded at 2 h post infection (h.p.i.). MjRad23 consists of putative functional domains including one ubiquitin domain (UBQ), two ubiquitin‐associated domains (UBA) and one heat‐shock chaperonin‐binding motif (STI1). Multiple alignment of MjRad23 with Rad23 of other species showed highly significant identity ranging from 37 to 53%; however, high homology is observed with Rad23 of Bombyx mori (BmRad23). UBQ domain region alignment revealed maximum of 66% homology with Rad23 of Apis melifera (AmRad23). MjRad23 clustered with invertebrate sector along with insect species in evolution analysis. Three‐dimensional structural analyses demonstrated the highest identity between MjRad23 and human Rad23A (hHR23A). Conclusions: The present work revealed the presence of MjRad23 gene, which is essential in DNA repair process. Further studies are required to clarify the involvement of MjRad23 in NER process. Significance and Impact of the Study: This is the first report on identification and characterization of DNA repair protein in crustaceans, which will lead to further investigation to explore the molecular mechanisms behind the NER process.  相似文献   

12.
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.  相似文献   

13.
Nucleotide excision repair (NER) in eukaryotes removes DNA base damage as an oligonucleotide in a complex series of reactions. The nature of the dual incision reactions on either side of the damaged base has been extensively investigated. However, the precise mechanism of cleavage of the phosphodiester backbone of the DNA by the NER endonucleases and how this relates to removal of the damage-containing oligonucleotide during the excision process has not been determined. We previously isolated a stable heterotrimeric complex of Rad7/Rad16/Abf1 from yeast which functions in the conserved global genome repair (GGR) pathway. GGR removes lesions from DNA that is not actively transcribing. We have shown previously that the Rad7/Rad16/Abf1 heterotrimer is required to observe DNA repair synthesis and oligonucleotide excision during in vitro NER, but not needed to detect NER-dependent incision in such reactions. Here we report that this protein complex generates superhelicity in DNA through the catalytic activity of the Rad16 component. The torsion generated in the DNA by this complex is necessary to remove the damage-containing oligonucleotide during NER--a process referred to as excision. We conclude that in yeast the molecular mechanism of NER includes the generation of superhelical torsion in DNA.  相似文献   

14.
15.
Nucleotide excision repair in yeast   总被引:22,自引:0,他引:22  
  相似文献   

16.
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.  相似文献   

17.
18.
Centrin/Cdc31 is a novel regulator of protein degradation   总被引:1,自引:0,他引:1  
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.  相似文献   

19.
Saccharomyces cerevisiae RAD4, RAD7, RAD16, and RAD23 genes function in the nucleotide excision repair (NER) of ultraviolet light (UV)-damaged DNA. Previous biochemical studies have shown that the Rad4 and Rad23 proteins are associated in a stoichiometric complex named NEF2, and the Rad7 and Rad16 proteins form another stoichiometric complex named NEF4. While NEF2 is indispensable for the incision of UV-damaged DNA in the in vitro reconstituted system, NEF4 stimulates the incision reaction. Both NEF2 and NEF4 bind UV-damaged DNA, which raises the intriguing possibility that these two complexes cooperate to achieve the high degree of specificity for DNA damage demarcation required for nucleotide excision repair in vivo. Consistent with this hypothesis, we find that NEF2 and NEF4 bind in a synergistic fashion to UV-damaged DNA in a reaction that is dependent on ATP. We also purify the Rad7 protein and show that it binds DNA but has no preference for UV-damaged DNA. Rad7 physically interacts with NEF2, suggesting a role for Rad7 in linking NEF2 with NEF4.  相似文献   

20.
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