A formamidopyrimidine derivative from the deoxyguanosine adduct produced by food contaminant acrylamide induces DNA replication block and mutagenesis.

Acrylamide, a common food contaminant, is metabolically activated to glycidamide, which reacts with DNA at the N7 position of dG, forming N7-(2-carbamoyl-2-hydroxyethyl)-dG (GA7dG). Owing to its chemical lability, the mutagenic potency of GA7dG has not yet been clarified. We found that GA7dG undergoes ring-opening hydrolysis to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine (GA-FAPy-dG), even at neutral pH. Therefore, we aimed to examine the effects of GA-FAPy-dG on the efficiency and fidelity of DNA replication using an oligonucleotide carrying GA-FAPy-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)guanine (dfG), a 2'-fluorine substituted analog of GA-FAPy-dG. GA-FAPy-dfG inhibited primer extension by both human replicative DNA polymerase ε and the translesion DNA synthesis polymerases (Polη, Polι, Polκ, and Polζ) and reduced the replication efficiency by less than half in human cells, with single base substitution at the site of GA-FAPy-dfG. Unlike other formamidopyrimidine derivatives, the most abundant mutation was G:C > A:T transition, which was decreased in Polκ- or REV1-KO cells. Molecular modeling suggested that a 2-carbamoyl-2-hydroxyethyl group at the N5 position of GA-FAPy-dfG can form an additional H-bond with thymidine, thereby contributing to the mutation. Collectively, our results provide further insight into the mechanisms underlying the mutagenic effects of acrylamide.

Thymine DNA glycosylase modulates DNA damage response and gene expression by base excision repair-dependent and independent mechanisms.

Thymine DNA glycosylase (TDG) is a base excision repair (BER) enzyme, which is implicated in correction of deamination-induced DNA mismatches, the DNA demethylation process and regulation of gene expression. Because of these pivotal roles associated, it is crucial to elucidate how the TDG functions are appropriately regulated in vivo. Here, we present evidence that the TDG protein undergoes degradation upon various types of DNA damage, including ultraviolet light (UV). The UV-induced degradation of TDG was dependent on proficiency in nucleotide excision repair and on CRL4CDT2 -mediated ubiquitination that requires a physical interaction between TDG and DNA polymerase clamp PCNA. Using the Tdg-deficient mouse embryonic fibroblasts, we found that ectopic expression of TDG compromised cellular survival after UV irradiation and repair of UV-induced DNA lesions. These negative effects on cellular UV responses were alleviated by introducing mutations in TDG that impaired its BER function. The expression of TDG induced a large-scale alteration in the gene expression profile independently of its DNA glycosylase activity, whereas a subset of genes was affected by the catalytic activity of TDG. Our results indicate the presence of BER-dependent and BER-independent functions of TDG, which are involved in regulation of cellular DNA damage responses and gene expression patterns.

Xeroderma pigmentosum group C protein interacts with histones: regulation by acetylated states of histone H3.

In the mammalian global genome nucleotide excision repair pathway, two damage recognition factors, XPC and UV-DDB, play pivotal roles in the initiation of the repair reaction. However, the molecular mechanisms underlying regulation of the lesion recognition process in the context of chromatin structures remain to be understood. Here, we show evidence that damage recognition factors tend to associate with chromatin regions devoid of certain types of acetylated histones. Treatment of cells with histone deacetylase inhibitors retarded recruitment of XPC to sites of UV-induced DNA damage and the subsequent repair process. Biochemical studies showed novel multifaceted interactions of XPC with histone H3, which were profoundly impaired by deletion of the N-terminal tail of histone H3. In addition, histone H1 also interacted with XPC. Importantly, acetylation of histone H3 markedly attenuated the interaction with XPC in vitro, and local UV irradiation of cells decreased the level of H3K27ac in the damaged areas. Our results suggest that histone deacetylation plays a significant role in the process of DNA damage recognition for nucleotide excision repair and that the localization and functions of XPC can be regulated by acetylated states of histones.

Stalled Polη at its cognate substrate initiates an alternative translesion synthesis pathway via interaction with REV1.

DNA polymerase η (Polη), whose gene mutation is responsible for the inherited disorder xeroderma pigmentosum variant (XP-V), carries out accurate and efficient translesion synthesis (TLS) across cyclobutane pyrimidine dimer (CPD). As Polη interacts with REV1, and REV1 interacts with other TLS polymerases including Polι, Polκ and Polζ, Polη may play a role in recruitment of these TLS polymerases at lesion site. But it is unclear whether UV sensitivity of XP-V patients is caused not only by defect of Polη activity but also by dysfunction of network between Polη and other TLS polymerases. Here, we examined whether the TLS polymerase network via Polη is important for replicative bypass of CPDs and DNA damage tolerance induced by UV in mouse cells. We observed that UV sensitivity of Polη-deficient mouse cells was moderately rescued by the expression of a catalytically inactive Polη. Moreover, this recovery of cellular UV sensitivity was mediated by the interaction between Polη and REV1. However, expression of the inactive mutant Polη was not able to suppress the incidence of UV-induced mutation observed in Polη-deficient cells. We propose the model that REV1 and Polκ are involved in DNA damage tolerance via Polη-REV1 interaction when Polη fails to bypass its cognate substrates.

Exploration of Trends in Antimicrobial Use and Their Determinants Based on Dispensing Information Collected from Pharmacies throughout Japan: A First Report.

The purpose of this study was to evaluate the defined daily doses (DDD)/1000 prescriptions/month (DPM) as a new indicator that can be used in pharmacies, and to describe antimicrobial use patterns in pharmacies nationwide in Japan. Dispensing volumes, number of prescriptions received, and facility information were obtained from 2638 pharmacies that participated in a survey. DPM was calculated based on the dispensing volume and number of prescriptions, which are routinely collected data that are simple to use. Use of third-generation cephalosporins, quinolones, and macrolides in pharmacies that received prescriptions primarily from hospitals or clinics decreased from January 2019 to January 2021. In particular, the antimicrobial use was higher in otorhinolaryngology departments than in other departments, despite a decrease in the antimicrobial use. In the linear multiple regression analysis, otorhinolaryngology department was independently associated with the third-generation cephalosporin, quinolone, and macrolide prescription in all periods. This study reveals for the first-time trends in antimicrobial use through a new indicator using the volume of drugs dispensed in pharmacies throughout Japan. Antimicrobial use differed by the medical department, suggesting the need to target interventions according to the department type.

Histone deacetylation regulates nucleotide excision repair through an interaction with the XPC protein.

The XPC protein complex plays a central role in DNA lesion recognition for global genome nucleotide excision repair (GG-NER). Lesion recognition can be accomplished in either a UV-DDB-dependent or -independent manner; however, it is unclear how these sub-pathways are regulated in chromatin. Here, we show that histone deacetylases 1 and 2 facilitate UV-DDB-independent recruitment of XPC to DNA damage by inducing histone deacetylation. XPC localizes to hypoacetylated chromatin domains in a DNA damage-independent manner, mediated by its structurally disordered middle (M) region. The M region interacts directly with the N-terminal tail of histone H3, an interaction compromised by H3 acetylation. Although the M region is dispensable for in vitro NER, it promotes DNA damage removal by GG-NER in vivo, particularly in the absence of UV-DDB. We propose that histone deacetylation around DNA damage facilitates the recruitment of XPC through the M region, contributing to efficient lesion recognition and initiation of GG-NER.

Comparison of Community Pharmacy Practice in Japan and US State of Illinois.

In 2006, a new 6-year educational system of pharmaceutical sciences was initiated to turn out strong clinical pharmacists in Japan. However, this new attempt is estimated not to fully satisfy the demand of clinical sites and the needs of the society in Japan. The objective of this study is to assess the performance of pharmaceutical services of community pharmacists in Illinois, United States, and Japan with the aim of comparing these services and barriers to pharmacy service delivery. The study designed as a cross-sectional, web-based study among US and Japan pharmacists. The survey asks several questions about demographic data, technical-related information and pharmaceutical services offered to patients, and pharmacy service performance. Almost 50 (92.6%) community pharmacists in United States reported that they dispensed more than 100 prescriptions in 1 day during the study period. In contrast, in Japan, community pharmacists (55.2%) dispensed 10 to 50 prescriptions during the same period. Half of the pharmacists in Japan either strongly agreed or agreed that they lack sufficient interpersonal and management skills. And many pharmacists agreed that lack of appropriate knowledge and insufficient training before graduation are major barriers to optimized pharmacy services in Japan. These findings can be used to promote discussion between Japanese pharmacists and stakeholders about pharmacy education programs in Japan and the future role of the community pharmacists in patient care in Japan.

Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts.

The (6-4) pyrimidine-pyrimidone photoproduct [(6-4)PP] is a major DNA lesion induced by ultraviolet radiation. (6-4)PP induces complex mutations opposite its downstream bases, in addition to opposite 3' or 5' base, as has been observed through a site-specific translesion DNA synthesis (TLS) assay. The mechanism by which these mutations occur is not well understood. To elucidate the mechanisms underlying mutagenesis induced by (6-4)PP, we performed an intracellular TLS assay using a replicative vector with site-specific T(thymidine)-T (6-4)PP. Rev3-/-p53-/- mouse embryonic fibroblast (MEF) cells (defective in Polζ) were almost completely defective in bypassing T-T (6-4)PP, whereas both Rev1-/- and Polh-/-Poli-/-Polk-/- MEF cells (defective in Polη, Polι, and Polκ) presented bypassing activity comparable to that of wild-type cells, indicating that Y-family TLS polymerases are dispensable for bypassing activity, whereas Polζ plays an essential role, probably at the extension step. Among all cells tested, misincorporation occurred most frequently just beyond the lesion (position +1), indicating that the Polζ-dependent extension step is crucial for (6-4)PP-induced mutagenesis. We then examined the effects of sequence context on T-T (6-4)PP bypass using a series of T-T (6-4)PP templates with different sequences at position +1 or -1 to the lesion, and found that the dependency of T-T (6-4)PP bypass on Polζ is not sequence specific. However, the misincorporation frequency at position +1 differed significantly among these templates. The misincorporation of A at position +1 occurred frequently when a purine base was located at position -1. These results indicate that Polζ-dependent extension plays a major role in inducing base substitutions in (6-4)PP-induced mutagenesis, and its fidelity is affected by sequence context surrounding a lesion.

Functional impacts of the ubiquitin-proteasome system on DNA damage recognition in global genome nucleotide excision repair.

The ubiquitin-proteasome system (UPS) plays crucial roles in regulation of various biological processes, including DNA repair. In mammalian global genome nucleotide excision repair (GG-NER), activation of the DDB2-associated ubiquitin ligase upon UV-induced DNA damage is necessary for efficient recognition of lesions. To date, however, the precise roles of UPS in GG-NER remain incompletely understood. Here, we show that the proteasome subunit PSMD14 and the UPS shuttle factor RAD23B can be recruited to sites with UV-induced photolesions even in the absence of XPC, suggesting that proteolysis occurs at DNA damage sites. Unexpectedly, sustained inhibition of proteasome activity results in aggregation of PSMD14 (presumably with other proteasome components) at the periphery of nucleoli, by which DDB2 is immobilized and sequestered from its lesion recognition functions. Although depletion of PSMD14 alleviates such DDB2 immobilization induced by proteasome inhibitors, recruitment of DDB2 to DNA damage sites is then severely compromised in the absence of PSMD14. Because all of these proteasome dysfunctions selectively impair removal of cyclobutane pyrimidine dimers, but not (6-4) photoproducts, our results indicate that the functional integrity of the proteasome is essential for the DDB2-mediated lesion recognition sub-pathway, but not for GG-NER initiated through direct lesion recognition by XPC.

Reevaluation of the role of DNA polymerase theta in somatic hypermutation of immunoglobulin genes.

DNA polymerase theta has been implicated in the process of somatic hypermutation in immunoglobulin variable genes based on several reports of alterations in the frequency and spectra of mutations from Polq(-/-) mice. However, these studies have contrasting results on mutation frequencies and the types of nucleotide substitutions, which question the role of polymerase theta in hypermutation. DNA polymerase eta has a dominant effect on mutation and may substitute in the absence of polymerase theta to affect the pattern. Therefore, we have examined mutation in mice deficient for both polymerases theta and eta. The mutation frequencies in rearranged variable genes from Peyer's patches were similar in wild type, Polq(-/-), Polh(-/-), and Polq(-/-)Polh(-/-) mice. The types of substitutions were also similar between wild type and Polq(-/-) clones, and between Polh(-/-) and Polq(-/-)Polh(-/-) clones. Furthermore, there was no difference in heavy chain class switching in splenic B cells from the four groups of mice. These results indicate that polymerase theta does not play a significant role in the generation of somatic mutation in immunoglobulin genes.

Genetic analysis reveals an intrinsic property of the germinal center B cells to generate A:T mutations.

The immunoglobulin genes undergo a high frequency of point mutations at both C:G and A:T pairs in the germinal center (GC) B cells. This hypermutation process is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. Replication of this lesion, or its repair intermediate the abasic site, could introduce C:G mutations but the mechanisms leading to mutations at non-damaged A:T pairs remain elusive. Using a lacZ-transgenic system in which endogenous genome mutations can be detected with high sensitivity, we found that GC B cells exhibited a much higher ratio of A:T mutations as compared to naïve B, non-GC B, and cells of other tissues. This property does not require AID or active transcription of the target gene, and is dependent on DNA polymerase eta. These in vivo results demonstrate that GC B cells are unique in having an intrinsic propensity to generate A:T mutations during repair of endogenous DNA damage. These findings have important implications in understanding how AID, which can only target C:G base pairs, is able to induce the entire spectrum of mutations observed in immunoglobulin variable region genes in GC B cells.

UV-B radiation induces epithelial tumors in mice lacking DNA polymerase eta and mesenchymal tumors in mice deficient for DNA polymerase iota.

DNA polymerase eta (Pol eta) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlight-induced skin cancer. We recently reported in a study of Polh mutant mice that Pol eta is involved in the somatic hypermutation of immunoglobulin genes, but the cancer predisposition of Polh-/- mice has not been examined until very recently. Another translesion synthesis polymerase, Pol iota, a Pol eta paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol iota is enigmatic. Here, we generated Polh Poli double-deficient mice and compared the tumor susceptibility of them with Polh- or Poli-deficient animals under the same genetic background. While Pol iota deficiency does not influence the UV sensitivity of mouse fibroblasts irrespective of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histological diagnosis after chronic treatment with UV light reveals that Pol iota deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not observed in Polh(-/-) mice. These results suggest the involvement of the Pol eta and Pol iota proteins in UV-induced skin carcinogenesis.

Mechanism and regulation of DNA damage recognition in nucleotide excision repair.

Nucleotide excision repair (NER) is a versatile DNA repair pathway, which can remove an extremely broad range of base lesions from the genome. In mammalian global genomic NER, the XPC protein complex initiates the repair reaction by recognizing sites of DNA damage, and this depends on detection of disrupted/destabilized base pairs within the DNA duplex. A model has been proposed that XPC first interacts with unpaired bases and then the XPD ATPase/helicase in concert with XPA verifies the presence of a relevant lesion by scanning a DNA strand in 5'-3' direction. Such multi-step strategy for damage recognition would contribute to achieve both versatility and accuracy of the NER system at substantially high levels. In addition, recognition of ultraviolet light (UV)-induced DNA photolesions is facilitated by the UV-damaged DNA-binding protein complex (UV-DDB), which not only promotes recruitment of XPC to the damage sites, but also may contribute to remodeling of chromatin structures such that the DNA lesions gain access to XPC and the following repair proteins. Even in the absence of UV-DDB, however, certain types of histone modifications and/or chromatin remodeling could occur, which eventually enable XPC to find sites with DNA lesions. Exploration of novel factors involved in regulation of the DNA damage recognition process is now ongoing.

Hypersensitivity of mouse embryonic fibroblast cells defective for DNA polymerases η, ι and κ to various genotoxic compounds: Its potential for application in chemical genotoxic screening.

Genotoxic agents cause modifications of genomic DNA, such as alkylation, oxidation, bulky adduct formation, and strand breaks, which potentially induce mutations and changes to the structure or number of genes. Majority of point mutations are generated during error-prone bypass of modified nucleotides (translesion DNA synthesis, TLS); however, when TLS fails, replication forks stalled at lesions eventually result in more lethal effects, formation of double-stranded breaks (DSBs). Here we compared sensitivities to various compounds among mouse embryonic fibroblasts derived from wild-type and knock-out mice lacking one of the three Y-family TLS DNA polymerases (Polη, Polι, and Polκ) or all of them (TKO). The compounds tested in this study include genotoxins such as methyl methanesulfonate (MMS) and nongenotoxins such as ammonium chloride. We found that TKO cells exhibited the highest sensitivities to most of the tested genotoxins, but not to the non-genotoxins. In order to quantitatively evaluate the hypersensitivity of TKO cells to different chemicals, we calculated ratios of half-maximal inhibitory concentration for WT and TKO cells. The ratios for 9 out of 10 genotoxins ranged from 2.29 to 5.73, while those for 5 nongenotoxins ranged from 0.81 to 1.63. Additionally, the two markers for DNA damage, ubiquitylated proliferating cell nuclear antigen and γ-H2AX after MMS treatment, were accumulated in TKO cells more greatly than in WT cells. Furthermore, following MMS treatment, TKO cells exhibited increased frequency of sister chromatid exchange compared with WT cells. These results indicated that the hypersensitivity of TKO cells to genotoxins resulted from replication fork stalling and subsequent DNA double-strand breaks, thus demonstrating that TKO cells should be useful for evaluating chemical genotoxicity.

Normal hypermutation in antibody genes from congenic mice defective for DNA polymerase iota.

Several low fidelity DNA polymerases participate in generating mutations in immunoglobulin genes. Polymerase eta is clearly involved in the process by causing substitutions of A:T base pairs, whereas polymerase iota has a controversial role. Although the frequency of mutations was decreased in the BL2 cell line deficient for polymerase iota, hypermutation was normal in the 129 strain of mice, which has a natural nonsense mutation in the Poli gene. It is possible that the mice compensated for the defect over time, or that polymerase eta substituted in the absence of polymerase iota. To examine polymerase iota in a genetically defined background, we backcrossed the 129 nonsense mutation to the C57BL/6 strain for six generations. Class switch recombination and hypermutation were studied in these mice and in congenic mice doubly deficient for both polymerases iota and eta. The absence of both polymerases did not affect production of IgG1, indicating that these enzymes are not involved in switch recombination. Poli(-/-F6) mice had the same types of nucleotide substitutions in variable genes as their C57BL/6 counterparts, and mice doubly deficient for polymerases iota and eta had the same mutational spectrum as Polh-/- mice. Thus, polymerase iota did not contribute to the mutational spectra, even in the absence of polymerase eta.

Signal transducer and activator of transcription 3 is a key regulator of keratinocyte survival and proliferation following UV irradiation.

UVB irradiation of signal transducer and activator of transcription 3 (Stat3)-deficient keratinocytes resulted in a high incidence of apoptosis compared with controls. Conversely, forced expression of Stat3 desensitized keratinocytes to UVB-induced apoptosis. Upon UVB exposure, keratinocyte Stat3 was rapidly dephosphorylated, followed by decreases of both Stat3 mRNA and protein levels in a p53-independent manner. Vanadate treatment reversed the UVB-induced down-regulation of Stat3 and generation of apoptotic keratinocytes, suggesting the involvement of a tyrosine phosphatase. Furthermore, Stat3 was required for UVB-induced proliferation of follicular keratinocytes, leading to epidermal thickening. Finally, constitutive activation of Stat3 was observed in UVB-induced squamous cell carcinomas of either mice or human origin. These data suggest that Stat3 is required for survival and proliferation of keratinocytes following UVB exposure and that Stat3 is tightly regulated as part of a novel protective mechanism against UVB-induced skin cancer.

Two mammalian homologs of yeast Rad23, HR23A and HR23B, as multifunctional proteins.

Mammalian cells express two homologs of yeast Rad23, the so-called homolog of Rad23 (HR23) proteins. The HR23 proteins were identified more than two decades ago as factors involved in initiation of global genome nucleotide excision repair (GG-NER) along with their interacting partner, xeroderma pigmentosum group C (XPC) protein. Because the HR23 genes encode proteins harboring ubiquitin-like (UBL) domains at their N-termini and two ubiquitin-associated (UBA) domains in their central- and C-terminal regions, the link between HR23 proteins and proteolytic degradation has been widely explored by several methods, including yeast two-hybrid screening and co-affinity purification. To date, various HR23 protein partners have been identified, and these proteins are involved not only in DNA repair, but also in ubiquitin-dependent protein degradation, transcriptional regulation, and cell cycle control. In addition, establishment of mouse strains lacking the HR23 genes and RNA silencing of these genes in human cells demonstrated their significance in animal development and cell growth. Through these studies, the functional differences between the two HR23 proteins have been gradually revealed. Furthermore, recent comprehensive proteomic analyses will help to elucidate the functional protein-protein networks involving the HR23 proteins.

Japanese Community Pharmacists' Dispensing Influences Medicine Price Reduction more than Prescription Numbers.

This study examined the economic efficiency of the separation of prescription and dispensation medicines between doctors in medical institutions and pharmacists in pharmacies. The separation system in Japanese prefectures was examined with publicly available data (Ministry of Health, Labour and Welfare, 2012-2014; retrieved from http://www.mhlw.go.jp/topics/medias/year). We investigated whether the separation system reduces the number of medicines or the medication cost of a prescription because of separating the economic management between prescribing and dispensing and the effect of mutual observation between doctors and pharmacists. It is optional for Japanese medical institutions to participate in the separation system. Consequently, the spreading rate of the separation system in each administrative district is highly variable. We examined the separation system effect using the National Healthcare Insurance data for three years, 2012-2014. We tested whether the separation system ratio for each prefecture was significantly correlated to the medication price or the number of medicines on a prescription. If spreading the separation system influenced the price of prescribed daily medications or the number of medicines, the correlation would be significant. As a result, the medication price was significantly negatively correlated with the separation system ratio, but the number of medicines was not significant. Therefore, the separation system was effective in reducing daily medication cost but had little influence on reducing the number of daily medicines. This was observed over three years in Japan.

Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage.

We have identified two fission yeast homologs of budding yeast Rad4 and human xeroderma pigmentosum complementation group C (XP-C) correcting protein, designated Rhp4A and Rhp4B. Here we show that the rhp4 genes encode NER factors that are required for UV-induced DNA damage repair in fission yeast. The rhp4A-deficient cells but not the rhp4B-deficient cells are sensitive to UV irradiation. However, the disruption of both rhp4A and rhp4B resulted in UV sensitivity that was greater than that of the rhp4A-deficient cells, revealing that Rhp4B plays a role in DNA repair on its own. Fission yeast has two pathways to repair photolesions on DNA, namely, nucleotide excision repair (NER) and UV-damaged DNA endonuclease-dependent excision repair (UVER). Studies with the NER-deficient rad13 and the UVER-deficient (Delta)uvde mutants showed the two rhp4 genes are involved in NER and not UVER. Assessment of the ability of the various mutants to remove cyclobutane pyrimidine dimers (CPDs) from the rbp2 gene locus indicated that Rhp4A is involved in the preferential repair of lesions on the transcribed DNA strand and plays the major role in fission yeast NER. Rhp4B in contrast acts as an accessory protein in non-transcribed strand (NTS) repair.

The carboxy-terminal domain of the XPC protein plays a crucial role in nucleotide excision repair through interactions with transcription factor IIH.

The xeroderma pigmentosum group C (XPC) protein specifically involved in genome-wide damage recognition for nucleotide excision repair (NER) was purified as a tight complex with HR23B, one of the two mammalian homologs of RAD23 in budding yeast. This XPC-HR23B complex exhibits strong binding affinity for single-stranded DNA, as well as preferential binding to various types of damaged DNA. To examine the structure-function relationship of XPC, a series of truncated mutant proteins were generated and assayed for various binding activities. The two domains participating in binding to HR23B and damaged DNA, respectively, were mapped within the carboxy-terminal half of XPC, which also contains an evolutionary conserved amino acid sequence homologous to the yeast RAD4 protein. We established that the carboxy-terminal 125 amino acids are dispensable for both HR23B and damaged DNA binding, while interactions with transcription factor IIH (TFIIH) are significantly impaired by truncation of this domain. Furthermore, deletion of the extreme carboxy-terminal domain totally abolished XPC activity in the cell-free NER reaction. These results suggest that following initial damage recognition, the carboxy terminus of XPC may be essential for the recruitment of TFIIH, and that most truncation mutations identified in XP-C patients result in non-functional proteins.