Structure of the XPC binding domain of hHR23A reveals hydrophobic patches for protein interaction

Rad23 proteins are involved both in the ubiquitin-proteasome pathway and in nucleotide excision repair (NER), but the relationship between these two pathways is not yet understood. The two human homologs of Rad23, hHR23A and B, are functionally redundant in NER and interact with xeroderma pigmentosum complementation group C (XPC) protein. The XPC-hHR23 complex is responsible for the specific recognition of damaged DNA, which is an early step in NER. The interaction of the XPC binding domain (XPCB) of hHR23A/B with XPC protein has been shown to be important for its optimal function in NER. We have determined the solution structure of XPCB of hHR23A. The domain consists of five amphipathic helices and reveals hydrophobic patches on the otherwise highly hydrophilic domain surface. The patches are predicted to be involved in interaction with XPC. The XPCB domain has limited sequence homology with any proteins outside of the Rad23 family except for sacsin, a protein involved in spastic ataxia of Charlevoix-Saguenay, which contains a domain with 35% sequence identity.     

Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome.

hHR23B is one of two human homologs of the Saccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that specifically complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro. Although a small proportion of hHR23B is tightly complexed with the XP-C responsible gene product, XPC protein, a vast majority exists as an XPC-free form, indicating that hHR23B has additional functions other than NER in vivo. Here we demonstrate that the human NER factor hHR23B as well as another human homolog of RAD23, hHR23A, interact specifically with S5a, a subunit of the human 26 S proteasome using the yeast two-hybrid system. Furthermore, hHR23 proteins were detected with S5a at the position where 26 S proteasome sediments in glycerol gradient centrifugation of HeLa S100 extracts. Intriguingly, hHR23B showed the inhibitory effect on the degradation of (125)I-lysozyme in the rabbit reticulocyte lysate. hHR23 proteins thus appear to associate with 26 S proteasome in vivo. From co-precipitation experiments using several series of deletion mutants, we defined the domains in hHR23B and S5a that mediate this interaction. From these results, we propose that part of hHR23 proteins are involved in the proteolytic pathway in cells.     

Stable binding of human XPC complex to irradiated DNA confers strong discrimination for damaged sites

Nucleotide excision repair (NER) of DNA damage requires an efficient means of discrimination between damaged and non-damaged DNA. Cells from humans with xeroderma pigmentosum group C do not perform NER in the bulk of the genome and are corrected by XPC protein, which forms a complex with hHR23B protein. This complex preferentially binds to some types of damaged DNA, but the extent of discrimination in comparison to other NER proteins has not been clear. Recombinant XPC, hHR23B, and XPC-hHR23B complex were purified. In a reconstituted repair system, hHR23B stimulated XPC activity tenfold. Electrophoretic mobility-shift competition measurements revealed a 400-fold preference for binding of XPC-hHR23B to UV damaged over non-damaged DNA. This damage preference is much greater than displayed by the XPA protein. The discrimination power is similar to that determined here in parallel for the XP-E factor UV-DDB, despite the considerably greater molar affinity of UV-DDB for DNA. Binding of XPC-hHR23B to UV damaged DNA was very fast. Damaged DNA-XPC-hHR23B complexes were stable, with half of the complexes remaining four hours after challenge with excess UV-damaged DNA at 30 degrees C. XPC-hHR23B had a higher level of affinity for (6-4) photoproducts than cyclobutane pyrimidine dimers, and some affinity for DNA treated with cisplatin and alkylating agents. XPC-hHR23B could bind to single-stranded M13 DNA, but only poorly to single-stranded homopolymers. The strong preference of XPC complex for structures in damaged duplex DNA indicates its importance as a primary damage recognition factor in non-transcribed DNA during human NER.     

Solution structure and backbone dynamics of the XPC-binding domain of the human DNA repair protein hHR23B

Human cells contain two homologs of the yeast RAD23 protein, hHR23A and hHR23B, which participate in the DNA repair process. hHR23B houses a domain (residues 277-332, called XPCB) that binds specifically and directly to the xeroderma pigmentosum group C protein (XPC) to initiate nucleotide excision repair (NER). This domain shares sequence homology with a heat shock chaperonin-binding motif that is also found in the stress-inducible yeast phosphoprotein STI1. We determined the solution structure of a protein fragment containing amino acids 275-342 of hHR23B (termed XPCB-hHR23B) and compared it with the previously reported solution structures of the corresponding domain of hHR23A. The periodic positioning of proline residues in XPCB-hHR23B produced kinked alpha helices and assisted in the formation of a compact domain. Although the overall structure of the XPCB domain was similar in both XPCB-hHR23B and XPCB-hHR23A, the N-terminal part (residues 275-283) of XPCB-hHR23B was more flexible than the corresponding part of hHR23A. We tried to infer the characteristics of this flexibility through (15)N-relaxation studies. The hydrophobic surface of XPCB-hHR23B, which results from the diverse distribution of N-terminal region, might give rise to the functional pleiotropy observed in vivo for hHR23B, but not for hHR23A.     

HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.

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.     

Centrin 2 stimulates nucleotide excision repair by interacting with xeroderma pigmentosum group C protein

Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome nucleotide excision repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed alpha-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.     

Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair

Nucleotide excision repair (NER) is carried out by xeroderma pigmentosum (XP) factors. Before the excision reaction, DNA damage is recognized by a complex originally thought to contain the XP group C responsible gene product (XPC) and the human homologue of Rad23 B (HR23B). Here, we show that centrin 2/caltractin 1 (CEN2) is also a component of the XPC repair complex. We demonstrate that nearly all XPC complexes contain CEN2, that CEN2 interacts directly with XPC, and that CEN2, in cooperation with HR23B, stabilizes XPC, which stimulates XPC NER activity in vitro. CEN2 has been shown to play an important role in centrosome duplication. Thus, those findings suggest that the XPC-CEN2 interaction may reflect coupling of cell division and NER.     

In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product

The initial step in mammalian nucleotide excision repair (NER) of the major UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), requires lesion recognition. It is believed that the heterodimeric proteins XPC/hHR23B and UV-DDB (UV-damaged DNA binding factor, composed of the p48 and p127 subunits) perform this function in genomic DNA, but their requirement and lesion specificity in vivo remains unknown. Using repair-deficient xeroderma pigmentosum (XP)-A cells that stably express photoproduct-specific photolyases, we determined the binding characteristics of p48 and XPC to either CPDs or 6-4PPs in vivo. p48 localized to UV-irradiated sites that contained either CPDs or 6-4PPs. However, XPC localized only to UV-irradiated sites that contained 6-4PPs, suggesting that XPC does not efficiently recognize CPDs in vivo. XPC did localize to CPDs when p48 was overexpressed in the same cell, signifying that p48 activates the recruitment of XPC to CPDs and may be the initial recognition factor in the NER pathway.     

Binding surface mapping of intra- and interdomain interactions among hHR23B,ubiquitin, and polyubiquitin binding site 2 of S5a

hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein. However, hHR23B and RAD23 also have many important functions related to general proteolysis. hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains. The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin. On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome. We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation. Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops. This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B. The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated. The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B. The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.     

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.     

Biochemical analysis of the damage recognition process in nucleotide excision repair

XPA, XPC-hHR23B, RPA, and TFIIH all are the damage recognition proteins essential for the early stage of nucleotide excision repair. Nonetheless, it is not clear how these proteins work together at the damaged DNA site. To get insight into the molecular mechanism of damage recognition, we carried out a comprehensive analysis on the interaction between damage recognition proteins and their assembly on damaged DNA. XPC physically interacted with XPA, but failed to stabilize the XPA-damaged DNA complex. Instead, XPC-hHR23B was effectively displaced from the damaged DNA by the combined action of RPA and XPA. A mutant RPA lacking the XPA interaction domain failed to displace XPC-hHR23B from damaged DNA, suggesting that XPA and RPA cooperate with each other to destabilize the XPC-hHR23B-damaged DNA complex. Interestingly, the presence of hHR23B significantly increased RPA/XPA-mediated displacement of XPC from damaged DNA, suggesting that hHR23B may modulate the binding of XPC to damaged DNA. Together, our results suggest that damage recognition occurs in a multistep process such that XPC-hHR23B initiates damage recognition, which was replaced by combined action of XPA and RPA. XPA and RPA, once forming a complex at the damage site, would likely work with TFIIH, XPG, and ERCC1-XPF for dual incision.     

Relative levels of the two mammalian Rad23 homologs determine composition and stability of the xeroderma pigmentosum group C protein complex

Mammalian cells express two Rad23 homologs, HR23A and HR23B, which have been implicated in regulation of proteolysis via the ubiquitin/proteasome pathway. Recently, the proteins have been shown to stabilize xeroderma pigmentosum group C (XPC) protein that is involved in DNA damage recognition for nucleotide excision repair (NER). Because the vast majority of XPC forms a complex with HR23B rather than HR23A, we investigated possible differences between the two Rad23 homologs in terms of their effects on the XPC protein. In wild-type mouse embryonic fibroblasts (MEFs), endogenous XPC was found to be relatively stable, while its steady-state level and stability appeared significantly reduced by targeted disruption of the mHR23B gene, but not by that of mHR23A. Loss of both mHR23 genes caused a strong further reduction of the XPC protein level. Quantification of the two mHR23 proteins revealed that in normal cells mHR23B is actually approximately 10 times more abundant than mHR23A. In addition, overexpression of mHR23A in the mHR23A/B double knock out cells restored not only the steady-state level and stability of the XPC protein, but also cellular NER activity to near wild-type levels. These results indicate that the two Rad23 homologs are largely functionally equivalent in NER, and that the difference in expression levels explains for a major part the difference in complex formation with as well as stabilization effects on XPC.     

Developmental Defects and Male Sterility in Mice Lacking the Ubiquitin-Like DNA Repair Gene mHR23B

mHR23B encodes one of the two mammalian homologs of Saccharomyces cerevisiae RAD23, a ubiquitin-like fusion protein involved in nucleotide excision repair (NER). Part of mHR23B is complexed with the XPC protein, and this heterodimer functions as the main damage detector and initiator of global genome NER. While XPC defects exist in humans and mice, mutations for mHR23A and mHR23B are not known. Here, we present a mouse model for mHR23B. Unlike XPC-deficient cells, mHR23B(-/-) mouse embryonic fibroblasts are not UV sensitive and retain the repair characteristics of wild-type cells. In agreement with the results of in vitro repair studies, this indicates that mHR23A can functionally replace mHR23B in NER. Unexpectedly, mHR23B(-/-) mice show impaired embryonic development and a high rate (90%) of intrauterine or neonatal death. Surviving animals display a variety of abnormalities, including retarded growth, facial dysmorphology, and male sterility. Such abnormalities are not observed in XPC and other NER-deficient mouse mutants and point to a separate function of mHR23B in development. This function may involve regulation of protein stability via the ubiquitin/proteasome pathway and is not or only in part compensated for by mHR23A.     

Structure-function analysis of the EF-hand protein centrin-2 for its intracellular localization and nucleotide excision repair

Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes.     

Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair

Human centrin 2 is a component of the nucleotide excision repair system, as a subunit of the heterotrimer including xeroderma pigmentosum group C protein (XPC) and hHR23B. The C-terminal domain of centrin (C-HsCen2) binds strongly a peptide from the XPC protein (P1-XPC: N(847)-R(863)). Here, we characterize the solution Ca(2+)-dependent structural and molecular features of the C-HsCen2 in complex with P1-XPC, mainly using NMR spectroscopy and molecular modeling. The N-terminal half of the peptide, organized as an alpha helix is anchored into a deep hydrophobic cavity of the protein, because of three bulky hydrophobic residues in position 1-4-8 and electrostatic contacts with the centrin helix E. Investigation of the whole centrin interactions shows that the N-terminal domain of the protein is not involved in the complex formation and is structurally independent from the peptide-bound C-terminal domain. The complex may exist in three different binding conformations corresponding to zero, one, and two Ca(2+)-bound states, which may exchange with various rates and have distinct structural stability. The various features of the intermolecular interaction presented here constitute a centrin-specific mode for the target binding.