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At least 19 recordsLinked to original sources
PubMed2005

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.

Amino Acid Motifs↗
PubMed1999

Functional studies on the interaction between human replication protein A and Xeroderma pigmentosum group A complementing protein (XPA).

The human replication protein A (RPA; also known as human single-stranded DNA binding protein, HSSB) is a multisubunit complex (70, 34 and 11 kDa subunits) involved in the three processes of DNA metabolism; replication, repair, recombination. We found that both 34 and 70 kDa subunits (p34 and p70, respectively), of RPA interacts with the Xeroderma pigmentosum group A complementing protein (XPA), a protein that specifically recognizes UV-damaged DNA. Our mutational analysis indicated that no particular domains of RPA p70 were essential for its interaction with XPA. We also examined the effect of this XPA-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the crude extract and monopolymerase system. XPA inhibited SV40 DNA replication in vitro through its interaction with RPA. Taken together, these results suggest that there is a role for RPA in the regulation of DNA metabolism through its ability to modulate the interactions of proteins involved in the processes of DNA metabolism.

Animals↗
PubMed1993

Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA.

The xeroderma pigmentosum group A complementing protein (XPAC) is involved in an early step of nucleotide excision repair, the main process that removes UV damage and many chemical lesions from DNA. To explore the properties and function of XPAC, recombinant protein encoded by the human XPAC cDNA was expressed with an N-terminal polyhistidine tag in Escherichia coli and purified to homogeneity. The soluble fusion protein could correct the repair defect in vitro of XP-A cell extracts. XPAC protein bound to DNA with a preference for UV-irradiated over nonirradiated DNA, as determined by a gel electrophoresis mobility shift assay with a 258 base pair DNA fragment (the association constant was approximately 3 x 10(6) M-1 for the fragment irradiated with 6 kJ/m2 UV light). Removal of cyclobutane pyrimidine dimers from UV-irradiated DNA by enzymatic photoreactivation did not significantly reduce binding of XPAC to the irradiated fragment, indicating that binding was mostly due to (6-4) photoproducts, with a preference for a (6-4) photoproduct over an undamaged base pair up to 300-fold. Undamaged single-stranded DNA competed about 4-fold more effectively than undamaged double-stranded DNA for binding of XPAC to a UV-irradiated fragment. In addition, XPAC bound to DNA treated with the chemotherapeutic agent cis-diamminedichloroplatinum(II). The results suggest that XPAC functions as a key component in recognition of DNA damage during repair.

Base Sequence↗
PubMed2006

The structure of the human centrin 2-xeroderma pigmentosum group C protein complex.

Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered alpha-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an alpha-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.

Amino Acid Motifs↗
PubMed2002

Strand-specific binding of RPA and XPA to damaged duplex DNA.

The nucleotide excision repair (NER) pathway is a major pathway used to repair bulky adduct DNA damage. Two proteins, xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA), have been implicated in the role of DNA damage recognition in the NER pathway. The particular manner in which these two damage recognition proteins align themselves with respect to a damaged DNA site was assessed using photoreactive base analogues within specific DNA substrates to allow site-specific cross-linking of the damage recognition proteins. Results of these studies demonstrate that both RPA and XPA are in close proximity to the adduct as measured by cross-linking of each protein directly to the platinum moiety. Additional studies demonstrate that XPA contacts both the damaged and undamaged strands of the duplex DNA. Direct evidence is presented demonstrating preferential binding of RPA to the undamaged strand of a duplex damaged DNA molecule.

Cisplatin↗
PubMed1996

Truncated XPA protein detected in atypical group A xeroderma pigmentosum.

XPA protein from a patient with typical group A xeroderma pigmentosum (XP) and three atypical group-A XP patients were analysed. Immunoblot analysis of XPA proteins revealed that a typical group-A XP patient showed no XPA protein band, while a smaller, truncated XPA protein, which appears to be responsible for mid skin lesions and minimal neurological abnormalities, was detected in cells from three atypical group-A XP patients. Furthermore, the difference in the amount of truncated XPA protein correlated with the mildness of neurological manifestations in these three atypical group-A XP patients. The results suggest a correlation between clinical manifestations and qualitative and quantitative abnormalities of XPA protein products.

Adolescent↗
PubMed2006

The novel tumor suppressor p33ING2 enhances nucleotide excision repair via inducement of histone H4 acetylation and chromatin relaxation.

p33ING2 is a novel candidate tumor suppressor, which has been shown to be involved in the regulation of gene transcription, cell cycle arrest, and apoptosis in a p53-dependent manner for maintaining the genomic stability. Previously, we showed that p33ING2 promoted UV-induced apoptosis in human melanoma cells. To further reveal the role of p33ING2 in cellular stress response to UV irradiation, we hypothesized that p33ING2 may enhance the repair of UV-damaged DNA, similarly to its homologue p33(ING1b). Using the host-cell reactivation assay, we show that overexpression of p33ING2 significantly enhances nucleotide excision repair of UV-induced DNA damage in melanoma cells in a p53-dependent manner. Furthermore, DNA repair is completely abolished in cells treated with p33ING2 small interfering RNA, suggesting that a physiologic level of p33ING2 is required for nucleotide excision repair. In addition, we found that p33ING2 is an essential factor for UV-induced rapid histone H4 acetylation, chromatin relaxation, and the recruitment of damage recognition protein, xeroderma pigmentosum group A protein, to the photolesions. These observations suggest that p33ING2 is required for the initial DNA damage sensing and chromatin remodeling in the nucleotide excision repair process.

Acetylation↗
PubMed2004

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.

Animals↗
PubMed2000

The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA.

The xeroderma pigmentosum group C protein complex XPC-HR23B was first isolated as a factor that complemented nucleotide excision repair defects of XP-C cell extracts in vitro. Recent studies have revealed that this protein complex plays an important role in the early steps of global genome nucleotide excision repair, especially in damage recognition, open complex formation, and repair protein complex formation. However, the precise function of XPC-HR23B in global genome repair is still unclear. Here we demonstrate that XPC-HR23B interacts with general transcription factor IIH (TFIIH) both in vivo and in vitro. This interaction is thought to be mediated through the specific affinity of XPC for the TFIIH subunits XPB and/or p62, which are essential for both basal transcription and nucleotide excision repair. Interestingly, association of TFIIH with DNA was observed in both wild-type and XP-A cell extracts but not in XP-C cell extracts, and XPC-HR23B could restore the association of TFIIH with DNA in XP-C cell extracts. Moreover, we found that XPC-HR23B was necessary for efficient association of TFIIH with damaged DNA in cell-free extracts. We conclude that the XPC-HR23B protein complex plays a crucial role in the recruitment of TFIIH to damaged DNA in global genome repair.

Cell Line↗
PubMed2003

Xeroderma pigmentosum group a protein and chemotherapy resistance in human germ cell tumors.

The exceptional sensitivity of germ cell tumors (GCTs) of adolescents and adults to chemotherapy, in particular to cisplatin, has been attributed to low levels of xeroderma pigmentosum group A protein (XPA), a crucial component of the nucleotide excision repair DNA repair pathway. In different types of solid tumors, resistance to cisplatin has been associated with enhanced expression of XPA. To assess the role of XPA levels in clinical sensitivity and resistance of GCTs to chemotherapy, immunohistochemistry was performed on tumor samples of both unselected patients before therapy and patients with fully documented clinical course before and after therapy. In the case of high XPA levels, fluorescent in situ hybridization was applied to assess the possibility of gene amplification. XPA protein levels were investigated by Western blot analysis after repeated exposure to cisplatin in different GCT-derived cell lines. Finally, XPA levels of both sensitive and cisplatin-resistant GCT cell lines were compared with cell lines derived from other neoplasms. We found that the presence of XPA protein as assessed by immunohistochemistry differs among the various histologies of GCTs. It is found more frequently and with a more homogenous staining pattern in histologic subtypes showing a more differentiated phenotype. Overall, no differences in the presence of XPA was observed between samples of tumors refractory or sensitive to chemotherapy. No XPA gene amplification was found. Interestingly, all tumors resected in relapse after chemotherapy in the refractory group stained positive for XPA. However, XPA was not induced by repeated courses of sublethal doses of cisplatin in GCT-derived cell lines in vitro, and no correlation between XPA protein levels and sensitivity to cisplatin in three GCT-derived cell lines was observed. We therefore conclude that XPA does not play a critical role in overall treatment resistance of GCTs.

Adolescent↗
PubMed2006

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.

Amino Acid Motifs↗
PubMed2003

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein.

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry.

Acetoxyacetylaminofluorene↗
PubMed2003

Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions.

Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair.

Cell Line↗
PubMed1995

Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication.

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase alpha-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA.

DNA Helicases↗
PubMed1994

Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23.

Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer-prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non-transcribing sequences ('genome-overall' repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP-C defect in a cell-free repair system containing UV-damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N-terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin-like N-terminus. The nature of the XP-C defect implies that the complex exerts a unique function in the genome-overall repair pathway which is important for prevention of skin cancer.

Amino Acid Sequence↗
PubMed1999

BCR binds to the xeroderma pigmentosum group B protein.

The BCR gene is involved in the formation of the BCR-ABL oncogene responsible for the pathogenesis of Philadelphia chromosome-positive human leukemias. We have previously shown that P210 BCR-ABL binds to the xeroderma pigmentosum group B protein (XPB) through the portion of BCR that is homologous to the catalytic domain of GDP-GTP exchangers such as yeast CDC24 and Dbl. In the baculovirus overexpression system which facilitates binding of coexpressed proteins, we now show that XPB binds to the intact BCR protein efficiently but not to CDC24 or Dbl, suggesting specificity of this interaction. The binding of endogenous BCR and XPB proteins was also detected in Hela cells, and this was inhibited by a blocking peptide. Full-length (1-782) XPB and its truncated form (203-782), which does not contain the nuclear localization signal, were tagged with glutathione S-transferase (GST) and were expressed in Rat1 fibroblasts. GST-XPB(203-782) was localized predominantly in the cytoplasm and bound to BCR but not to p62, one of the other components in TFIIH. GST-XPB(1-782) was largely in the nucleus and bound to p62 and BCR. Although the biological significance of the binding remains to be uncovered, BCR binds to the XPB/p62 complex.

Animals↗
PubMed2005

Thermodynamic properties of damaged DNA and its recognition by xeroderma pigmentosum group A protein and replication protein A.

The effects of the lesions induced by single, site-specific 1,2-GG or 1,3-GTG intrastrand adducts of cis-diamminedichloroplatinum(II) formed in oligodeoxyribonucleotide duplexes on energetics of DNA were examined by means of differential scanning calorimetry. These effects were correlated with affinity of these duplexes for damaged-DNA binding-proteins XPA and RPA; this affinity was examined by gel electrophoresis. The results confirm that rigid DNA bending is the specific determinant responsible for high-affinity interactions of XPA with damaged DNA, but that an additional important factor, which affects affinity of XPA to damaged DNA, is a change of thermodynamic stability of DNA induced by the damage. In addition, the results also confirm that RPA preferentially binds to DNA distorted so that hydrogen bonds between complementary bases are interrupted. RPA also binds to non-denaturational distortions in double-helical DNA, but affinity of RPA to these distortions is insensitive to alterations of thermodynamic stability of damaged DNA.

Base Sequence↗