Check, Check Triple Check: Multi-Step DNA Lesion Identification by Nucleotide Excision Repair.

In this issue, Li et al. (2015) uncover roles for the XPB and XPD helicases and for XPA during damage verification in nucleotide excision repair, supporting a novel tripartite damage checking mechanism that combines extreme versatility with narrow specificity.

Tripartite DNA Lesion Recognition and Verification by XPC, TFIIH, and XPA in Nucleotide Excision Repair.

Transcription factor IIH (TFIIH) is essential for both transcription and nucleotide excision repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten-subunit TFIIH and its seven-subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non-genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity in the presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC-dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.

Potential risk of esophageal squamous cell carcinoma due to nucleotide excision repair XPA and XPC gene variants and their interaction among themselves and with environmental factors.

The association of nucleotide excision repair (NER) gene polymorphisms with esophageal squamous cell carcinoma (ESCC) is inconclusive. The aim of the current study was to assess the association of repair gene xeroderma pigmentosum A (XPA) (rs-1800975) and xeroderma pigmentosum C (XPC) (rs-2228000) polymorphisms with ESCC risk as well as modifying effects of environmental factors. The genotyping was done in 450 confirmed ESCC cases and equal number of individually matched controls by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods. Conditional logistic regression models were used to assess the genotypic associations and interactions. A high ESCC risk was found in subjects who carried the homozygous minor allele of XPA (odds ratio (OR) = 3.57; 95 % confidence interval (CI) = 1.76-7.23), and the risk was higher when analysis was limited to participants who were ever smokers (OR = 4.22; 95 % CI = 2.01-8.88), lived in adobe houses (OR = 8.42; 95 % CI = 3.74-18.95), consumed large volumes of salt tea (OR = 7.42; 95 % CI = 3.30-16.69), or had a positive family history of cancer (FHC) (OR = 9.47; 95 % CI = 4.67-19.20). In case of XPC, a homozygous minor allele also showed strong association with ESCC risk (OR = 4.43; 95 % CI = 2.41-8.16). We again observed a very strong effect of the above environmental factors in elevating the risk of ESCC. Further, the variant genotypes of both genes in combination showed an increased risk towards ESCC (OR = 7.01; 95 % CI = 3.14-15.64) and such association was synergistically significant. Salt tea consumption showed an interaction with genotypes of XPA and XPC. However, an interaction with FHC was significant in the case of XPA genotype only. XPA and XPC genotypes are associated with an increased risk of ESCC, and such association was reasonably modulated by different exposures.

Benzo[a]pyrene (BP) DNA adduct formation in DNA repair-deficient p53 haploinsufficient [Xpa(-/-)p53(+/-)] and wild-type mice fed BP and BP plus chlorophyllin for 28 days.

We have evaluated DNA damage (DNA adduct formation) after feeding benzo[a]pyrene (BP) to wild-type (WT) and cancer-susceptible Xpa(-/-)p53(+/-) mice deficient in nucleotide excision repair and haploinsufficient for the tumor suppressor p53. DNA damage was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS), which measures r7,t8,t9-trihydroxy-c-10-(N (2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG), and a chemiluminescence immunoassay (CIA), using anti-r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum, which measures both BPdG and the other stable BP-DNA adducts. When mice were fed 100 ppm BP for 28 days, BP-induced DNA damage measured in esophagus, liver and lung was typically higher in Xpa(-/-)p53(+/-) mice, compared with WT mice. This result is consistent with the previously observed tumor susceptibility of Xpa(-/-)p53(+/-) mice. BPdG, the major DNA adduct associated with tumorigenicity, was the primary DNA adduct formed in esophagus (a target tissue in the mouse), whereas total BP-DNA adducts predominated in higher levels in the liver (a non-target tissue in the mouse). In an attempt to lower BP-induced DNA damage, we fed the WT and Xpa(-/-)p53(+/-) mice 0.3% chlorophyllin (CHL) in the BP-containing diet for 28 days. The addition of CHL resulted in an increase of BP-DNA adducts in esophagus, liver and lung of WT mice, a lowering of BPdG in esophagi of WT mice and livers of Xpa(-/-)p53(+/-) mice and an increase of BPdG in livers of WT mice. Therefore, the addition of CHL to a BP-containing diet showed a lack of consistent chemoprotective effect, indicating that oral CHL administration may not reduce PAH-DNA adduct levels consistently in human organs.

Impaired spermatogenesis and elevated spontaneous tumorigenesis in xeroderma pigmentosum group A gene (Xpa)-deficient mice.

We have reported that xeroderma pigmentosum group A (Xpa) gene-knockout mice [Xpa (-/-) mice] are deficient in nucleotide excision repair (NER) and highly sensitive to UV-induced skin carcinogenesis. Although xeroderma pigmentosum group A patients show growth retardation, immature sexual development, and neurological abnormalities as well as a high incidence of UV-induced skin tumors, Xpa (-/-) mice were physiologically and behaviorally normal. In the present study, we kept Xpa (-/-) mice for 2 years under specific pathogen-free (SPF) conditions and found that the testis diminished in an age-dependent manner, and degenerating seminiferous tubules and no spermatozoa were detected in the 24-month-old Xpa (-/-) mice. In addition, a higher incidence of spontaneous tumorigenesis was observed in the 24-month-old Xpa (-/-) mice compared to Xpa (+/+) controls. Xpa (-/-) mice provide a useful model for investigating the aging and internal tumor formation in XPA patients.

Increased expression of p53 enhances transcription-coupled repair and global genomic repair of a UVC-damaged reporter gene in human cells.

Ultraviolet (UV) light-induced DNA damage is repaired by nucleotide excision repair, which is divided into two sub-pathways: global genome repair (GGR) and transcription-coupled repair (TCR). While it is well established that the GGR pathway is dependent on the p53 tumour suppressor protein in human cells, both p53-dependent and p53-independent pathways have been reported for TCR. In the present work, we investigated the role of p53 in both GGR and TCR of a UVC-damaged reporter gene in human fibroblasts. We employed a non-replicating recombinant human adenovirus, AdCA17lacZ, that can efficiently infect human fibroblasts and express the beta-galactosidase (beta-gal) reporter gene under the control of the human cytomegalovirus promoter. We examined host cell reactivation (HCR) of beta-gal expression for the UVC-treated reporter construct in normal fibroblasts and in xeroderma pigmentosum (XP) and Cockayne syndrome (CS) fibroblasts deficient in GGR, TCR, or both. HCR was examined in fibroblasts that had been pre-infected with Ad5p53wt, which expresses wild-type p53, or a control adenovirus, AdCA18luc, which expresses the luciferase gene. We show that increased expression of p53 results in enhanced HCR of the UVC-damaged reporter gene in both untreated and UVC-treated cells for normal, CS-B (TCR-deficient), and XP-C (GGR-deficient), but not XP-A (TCR- and GGR-deficient) fibroblasts. These results indicate an involvement of p53 in both TCR and GGR of the UV-damaged reporter gene in human cells.

XPA gene, its product and biological roles.

The 31 kDa XPA protein is part of the core incision complex of the mammalian nucleotide excision repair (NER) system and interacts with DNA as well as with many other NER subunits. In the absence of XPA, no incision complex can form and no excision of damaged DNA damage occurs. A comparative analysis of the DNA-binding properties in the presence of different substrate conformations indicated that XPA protein interacts preferentially with kinked DNA backbones. The DNA-binding domain of XPA protein displays a positively charged deft that is involved in an indirect readout mechanism, presumably by detecting the increased negative potential encountered at sharp DNA bends. We propose that this indirect recognition function contributes to damage verification by probing the susceptibility of the DNA substrate to be kinked during the assembly of NER complexes.

A role for polymerase eta in the cellular tolerance to cisplatin-induced damage.

Mutation of the POLH gene encoding DNA polymerase eta (pol eta) causes the UV-sensitivity syndrome xeroderma pigmentosum-variant (XP-V) which is linked to the ability of pol eta to accurately bypass UV-induced cyclobutane pyrimidine dimers during a process termed translesion synthesis. Pol eta can also bypass other DNA damage adducts in vitro, including cisplatin-induced intrastrand adducts, although the physiological relevance of this is unknown. Here, we show that independent XP-V cell lines are dramatically more sensitive to cisplatin than the same cells complemented with functional pol eta. Similar results were obtained with the chemotherapeutic agents, carboplatin and oxaliplatin, thus revealing a general requirement for pol eta expression in providing tolerance to these platinum-based drugs. The level of sensitization observed was comparable to that of XP-A cells deficient in nucleotide excision repair, a recognized and important mechanism for repair of cisplatin adducts. However, unlike in XP-A cells, the absence of pol eta expression resulted in a reduced ability to overcome cisplatin-induced S phase arrest, suggesting that pol eta is involved in translesion synthesis past these replication-blocking adducts. Subcellular localization studies also highlighted an accumulation of nuclei with pol eta foci that correlated with the formation of monoubiquitinated proliferating cell nuclear antigen following treatment with cisplatin, reminiscent of the response to UV irradiation and further indicating a role for pol eta in dealing with cisplatin-induced damage. Together, these data show that pol eta represents an important determinant of cellular responses to cisplatin, which could have implications for acquired or intrinsic resistance to this key chemotherapeutic agent.

Selective DNA damage responses in murine Xpa-/-, Xpc-/- and Csb-/- keratinocyte cultures.

Cellular DNA damage responses (DDRs) are induced by unrepaired DNA lesions and constitute a protective back-up system that prevents the expansion of damaged cells. These cellular signaling pathways trigger either growth arrest or cell death and are believed to be major components of an early anti-cancer barrier. Cultures of C57BL/6J keratinocytes with various defects in NER sub-pathways allowed us to follow the kinetics of DDRs in an isogenic background and in the proper (physiologically relevant) target cells, supplementing earlier studies in heterogenic human fibroblasts. In a series of well-controlled parallel experiments we have shown that, depending on the NER deficiency, murine keratinocytes elicited highly selective DDRs. After a dose of UV-B that did not affect wild-type keratinocytes, Xpa(-/-) keratinocytes (complete NER deficiency) showed a rapid depletion of DNA replicating S-phase cells, a transient increase in quiescent S-phase cells (not replicating DNA), followed by massive apoptosis. Csb(-/-) keratinocytes (TC-NER deficient) responded by a more sustained increase in QS-phase cells and appeared more resistant to UV-B induced apoptosis than Xpa(-/-). In irradiated Xpc(-/-) keratinocytes (GG-NER deficient) the loss of replicating S-phase cells was associated with a gradual build-up of both QS-phase cells and cells arrested in late-S phase, in complete absence of apoptosis. Our analysis complements and extends previous in vivo investigations and highlights both similarities and differences with earlier fibroblast studies. In vitro cultures of murine keratinocytes provide a new tool to unravel the molecular mechanisms of UV-induced cellular stress responses in great detail and in a physiologically relevant background. This will be essential to fully appreciate the implications of DDRs in tumor suppression and cancer prevention.

Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation.

Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER.

A Functional Polymorphism (rs10817938) in the XPA Promoter Region Is Associated with Poor Prognosis of Oral Squamous Cell Carcinoma in a Chinese Han Population.

Single nucleotide polymorphisms of XPA gene have been studied in several cancers such as rs10817938, rs2808668. However, the role of XPA polymorphisms in patients with oral squamous cell carcinoma (OSCC) remains unclear. Thus, we analyzed the association of XPA polymorphisms with OSCC risk, clinicopathological characteristics and prognosis in the present study. TaqMan genotyping was used to evaluate the frequency of rs10817938, rs2808668 polymorphisms in OSCC patients. The prognostic significance of these polymorphisms was evaluated using Kaplan-Meier curves, Log-Rank analyses, and the Cox proportional hazard model. Luciferase reporter assay, RT-PCR and western blot were used to determine whether rs10817938 could influence transcription activity and XPA expression. The results showed that individuals carrying TC and CC genotypes had significantly greater risk of developing OSCC (OR = 1.42, 95% CI 1.04-1.93; OR = 2.75, 95% CI 1.32-5.71, respectively) when compared with wild-type TT genotype at rs10817938. OSCC patients with C allele at rs10817938 were more susceptible to lymph metastases, poor pathological differentiation and late TNM stage (OR = 1.67, 95% CI 1.17-2.37; OR = 1.64, 95% CI 1.18-2.28; OR = 1.54, 95% CI 1.11-2.14; respectively). A significant gene-environment interaction between smoking and CC genotype at rs10817938 was observed (COR = 3.60, 95% CI 1.20-10.9) and data also showed that OSCC patients with CC genotype and C allele had worse survival (p<0.001 for both). The T to C substitution at rs10817938 significantly decreased transcription activity of XPA gene, XPA mRNA and protein were also decreased in individuals with C allele at rs10817938. In addition, no significant association of rs2808668 polymorphism with OSCC risk, prognosis could be observed. In conclusion, the present study showed that XPA rs10817938 polymorphism is a functional SNP in vitro and in vivo and a biomarker for poor prognosis in OSCC patients.

TP53 mutations induced by BPDE in Xpa-WT and Xpa-Null human TP53 knock-in (Hupki) mouse embryo fibroblasts.

Somatic mutations in the tumour suppressor gene TP53 occur in more than 50% of human tumours; in some instances exposure to environmental carcinogens can be linked to characteristic mutational signatures. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalization assay (HIMA) is a useful model for studying the impact of environmental carcinogens on TP53 mutagenesis. In an effort to increase the frequency of TP53-mutated clones achievable in the HIMA, we generated nucleotide excision repair (NER)-deficient HUFs by crossing the Hupki mouse with an Xpa-knockout (Xpa-Null) mouse. We hypothesized that carcinogen-induced DNA adducts would persist in the TP53 sequence of Xpa-Null HUFs leading to an increased propensity for mismatched base pairing and mutation during replication of adducted DNA. We found that Xpa-Null Hupki mice, and HUFs derived from them, were more sensitive to the environmental carcinogen benzo[a]pyrene (BaP) than their wild-type (Xpa-WT) counterparts. Following treatment with the reactive metabolite of BaP, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), Xpa-WT and Xpa-Null HUF cultures were subjected to the HIMA. A significant increase in TP53 mutations on the transcribed strand was detected in Xpa-Null HUFs compared to Xpa-WT HUFs, but the TP53-mutant frequency overall was not significantly different between the two genotypes. BPDE induced mutations primarily at G:C base pairs, with approximately half occurring at CpG sites, and the predominant mutation type was G:C>T:A in both Xpa-WT and Xpa-Null cells. Further, several of the TP53 mutation hotspots identified in smokers' lung cancer were mutated by BPDE in HUFs (codons 157, 158, 245, 248, 249, 273). Therefore, the pattern and spectrum of BPDE-induced TP53 mutations in the HIMA are consistent with TP53 mutations detected in lung tumours of smokers. While Xpa-Null HUFs exhibited increased sensitivity to BPDE-induced damage on the transcribed strand, NER-deficiency did not enhance TP53 mutagenesis resulting from damage on the non-transcribed strand in this model.

Oxidative DNA damage and nucleotide excision repair.

SIGNIFICANCE: Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Accumulating evidence supports the hypothesis that nucleotide excision repair (NER), besides base excision repair (BER), is also involved in neutralizing oxidative DNA damage. RECENT ADVANCES: NER includes two distinct sub-pathways: transcription-coupled NER (TC-NER) and global genome repair (GG-NER). The CSA and CSB proteins initiate the onset of TC-NER. Recent findings show that not only CSB, but also CSA is involved in the repair of oxidative DNA lesions, in the nucleus as well as in mitochondria. The XPG protein is also of importance for the removal of oxidative DNA lesions, as it may enhance the initial step of BER. Substantial evidence exists that support a role for XPC in NER and BER. XPC deficiency not only results in decreased repair of oxidative lesions, but has also been linked to disturbed redox homeostasis. CRITICAL ISSUES: The role of NER proteins in the regulation of the cellular response to oxidative (mitochondrial and nuclear) DNA damage may be the underlying mechanism of the pathology of accelerated aging in Cockayne syndrome patients, a driving force for internal cancer development in XP-A and XP-C patients, and a contributor to the mixed exhibited phenotypes of XP-G patients. FUTURE DIRECTIONS: Accumulating evidence indicates that DNA repair factors can be involved in multiple DNA repair pathways. However, the distinct detailed mechanism and consequences of these additional functions remain to be elucidated and can possibly shine a light on clinically related issues.

Slow accumulation of mutations in Xpc-/- mice upon induction of oxidative stress.

XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa(-/-) and Xpc(-/-) exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc(-/-) mice, in contrary to Xpa(-/-) and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc(-/-) mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.

Both XPA and DNA polymerase eta are necessary for the repair of doxorubicin-induced DNA lesions.

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA.

[Effect of XPA expression on the chemotherapy sensitivity of A549/DDP cells].

AIM: To investigate the influence on platinum-based chemotherapy sensitivity by silencing xeroderma pigmentosum group A (XPA) gene expression in non-small cell lung cancer (NSCLC) drug resistance cell lines (A549/DDP). METHODS: We detected the expression of XPA in lung normal and tumor tissues by immunohistochemistry, quantitative real-time PCR (qPCR) and Western blotting. We silenced XPA expression in A549/DDP cells by XPA-shRNA transfection, and detected the expression of XPA by qPCR and Western blotting. The cell sensitivity to cisplatin and the apoptosis of A549/DDP cells transfected with XPA-shRNA were determined by MTT assay. RESULTS: The expression of XPA was higher in NSCLC tissues than that in normal lung tissues. Silencing XPA gene increased the apoptosis and sensitivity of A549/DDP cells to cisplatin. CONCLUSION: Silencing XPA gene can partly reverse the cisplatin resistance in human cisplatin-resistant NSCLC cell line A549/DDP.

The cross talk between pathways in the repair of 8-oxo-7,8-dihydroguanine in mouse and human cells.

Although oxidatively damaged DNA is repaired primarily via the base excision repair (BER) pathway, it is now evident that multiple subpathways are needed. Yet, their relative contributions and coordination are still unclear. Here, mouse embryo fibroblasts (MEFs) from selected nucleotide excision repair (NER) and/or BER mouse mutants with severe (Csb(m/m)/Xpa(-/-) and Csb(m/m)/Xpc(-/-)), mild (Csb(m/m)), or no progeria (Xpa(-/-), Xpc(-/-), Ogg1(-/-), Csb(m/m)/Ogg1(-/-)) or wild-type phenotype were exposed to an oxidizing agent, potassium bromate, and genomic 8-oxo-7,8-dihydroguanine (8-oxoGua) levels were measured by HPLC-ED. The same oxidized DNA base was measured in NER/BER-defective human cell lines obtained after transfection with replicative plasmids encoding siRNA targeting DNA repair genes. We show that both BER and NER factors contribute to the repair of 8-oxoGua, although to different extents, and that the repair profiles are similar in human compared to mouse cells. The BER DNA glycosylase OGG1 dominates 8-oxoGua repair, whereas NER (XPC, XPA) and transcription-coupled repair proteins (CSB and CSA) are similar, but minor contributors. The comparison of DNA oxidation levels in double versus single defective MEFs indicates increased oxidatively damaged DNA only when both CSB and XPC/XPA are defective, indicating that these proteins operate in different pathways. Moreover, we provide the first evidence of an involvement of XPA in the control of oxidatively damaged DNA in human primary cells.

Proanthocyanidins inhibit photocarcinogenesis through enhancement of DNA repair and xeroderma pigmentosum group A-dependent mechanism.

Dietary grape seed proanthocyanidins (GSP) inhibit photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. As ultraviolet B (UVB)-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) has been implicated in skin cancer risk, we studied whether dietary GSPs enhance repair of UVB-induced DNA damage and, if so, what is the potential mechanism? Supplementation of GSPs (0.5%, w/w) with AIN76A control diet significantly reduced the levels of CPD(+) cells in UVB-exposed mouse skin; however, GSPs did not significantly reduce UVB-induced CPD(+) cells in the skin of interleukin-12p40 (IL-12) knockout (KO) mice, suggesting that IL-12 is required for the repair of CPDs by GSPs. Using IL-12 KO mice and their wild-type counterparts and standard photocarcinogenesis protocol, we found that supplementation of control diet with GSPs (0.5%, w/w) significantly reduced UVB-induced skin tumor development in wild-type mice, which was associated with the elevated mRNA levels of nucleotide excision repair genes, such as XPA, XPC, DDB2, and RPA1; however, this effect of GSPs was less pronounced in IL-12 KO mice. Cytostaining analysis revealed that GSPs repaired UV-induced CPD(+) cells in xeroderma pigmentosum complementation group A (XPA)-proficient fibroblasts from a healthy individual but did not repair in XPA-deficient fibroblasts from XPA patients. Furthermore, GSPs enhance nuclear translocation of XPA and enhanced its interactions with other DNA repair protein ERCC1. Together, our findings reveal that prevention of photocarcinogenesis by GSPs is mediated through enhanced DNA repair in epidermal cells by IL-12- and XPA-dependent mechanisms.

Domains in the XPA protein important in its role as a processivity factor.

XPA is a protein essential for nucleotide excision repair (NER) where it is thought to function in damage recognition/verification. We have proposed an additional role, that of a processivity factor, conferring a processive mechanism of action on XPF and XPG, the endonucleases, involved in NER. The present study was undertaken to examine the domain(s) in the XPA gene that are important for the ability of the XPA protein to function as a processivity factor. Using site-directed mutagenesis, mutations were created in several of the exons of XPA and mutant XPA proteins produced. The results showed that the DNA binding domain of XPA is critical for its ability to act as a processivity factor. Mutations in both the zinc finger motif and the large basic cleft in this domain eliminated the ability of XPA to confer a processive mechanism of action on the endonucleases involved in NER.

Conditions of endoplasmic reticulum stress favor the accumulation of cytosolic prion protein.

After signal sequence-dependent targeting to the endoplasmic reticulum (ER), prion protein (PrP) undergoes several post-translational modifications, including glycosylation, disulfide bond formation, and the addition of a glycosylphosphatidylinositol anchor. As a result, multiple isoforms are generated. Because of the intrinsic weakness of the PrP signal sequence, a fraction of newly synthesized molecules fails to translocate and localizes to the cytosol. The physiopathologic role of this cytosolic isoform is still being debated. Here we have shown that, in both cultured cell lines and primary neurons, ER stress conditions weaken PrP co-translational translocation, favoring accumulation of aggregation-prone cytosolic species, which retain the signal sequence but lack N-glycans and disulfides. Inhibition of proteasomes further increases the levels of cytosolic PrP. Overexpression of spliced XBP1 facilitates ER translocation, suggesting that downstream elements of the Ire1-XBP1 pathway are involved in PrP targeting. These studies reveal a link between ER stress and the formation of cytosolic PrP isoforms potentially endowed with novel signaling or cytotoxic functions.