[Show abstract][Hide abstract] ABSTRACT: Mutations in XPD cause xeroderma pigmentosum (XP), XP and Cockayne syndrome (CS) crossover syndrome (XP/CS), trichothiodystrophy and cerebro-oculo-facio-skeletal syndrome (COFS). COFS represents the most severe end of the CS spectrum. This study reports two Japanese patients, COFS-05-135 and COFS-Chiba1, who died at ages of <1 year and exhibited typical COFS manifestations caused by XPD mutations p.[I619del];[R666W] and p.[G47R];[I619del], respectively. Two other cases of severe XP-D/CS (XP group D/CS), XP1JI (p.[G47R];) and XPCS1PV (p.[R666W];), died at ages <2 years. On the other hand, two cases of mild XP-D/CS, XP1NE (p.[G47R];[L461V;V716_R730del]) and XPCS118LV (p.[L461V;V716_R730del];[R666W]), lived beyond 37 years of age. p.I619Del and p.[L461V;V716_R730del] are functionally null; therefore, despite the differences in clinical manifestations, the functional protein in all of these patients was either p.G47R or p.R666W. To resolve the discrepancies in these XPD genotype-phenotype relationships, the p.[L461V;V716_R730del] allele was analyzed and we found that p.[L461V;A717G] was expressed from the same allele as p.[L461V;V716_R730del] by authentic splicing. Additionally, p.[L461V;A717G] could partially rescue the loss of XPD function, resulting in the milder manifestations observed in XP1NE and XPCS118LV.Journal of Human Genetics advance online publication, 26 February 2015; doi:10.1038/jhg.2015.18.
Journal of Human Genetics 02/2015; 60(5). DOI:10.1038/jhg.2015.18 · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Histone H2A variant H2AX is phosphorylated at Ser139 in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0-phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1 and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical non-proliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB.
[Show abstract][Hide abstract] ABSTRACT: The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur assembly (CIA) factors form a complex and have central roles in CIA assembly. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein, IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18 and CIAO1 form a tight ″core″ complex and IOP1 is an ″external″ component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to downregulation of all of the components. By contrast, IOP1 knockdown does not affect the level of any core component. In MMS19 overproducing cells, other core components are also upregulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in CIA reaction such as Fe-S helicases and the core complex might participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any cases, IOP1 functions in MMS19 dependent CIA pathway. We also revealed that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.
[Show abstract][Hide abstract] ABSTRACT: Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome.
DNA repair 04/2012; 11(5):488-501. DOI:10.1016/j.dnarep.2012.02.004 · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: UV-sensitive syndrome (UV(S)S) is an autosomal recessive disorder characterized by photosensitivity and deficiency in transcription-coupled repair (TCR), a subpathway of nucleotide-excision repair that rapidly removes transcription-blocking DNA damage. Cockayne syndrome is a related disorder with defective TCR and consists of two complementation groups, Cockayne syndrome (CS)-A and CS-B, which are caused by mutations in ERCC8 (CSA) and ERCC6 (CSB), respectively. UV(S)S comprises three groups, UV(S)S/CS-A, UV(S)S/CS-B and UV(S)S-A, caused by mutations in ERCC8, ERCC6 and an unidentified gene, respectively. Here, we report the cloning of the gene mutated in UV(S)S-A by microcell-mediated chromosome transfer. The predicted human gene UVSSA (formerly known as KIAA1530)(7) corrects defective TCR in UV(S)S-A cells. We identify three nonsense and frameshift UVSSA mutations in individuals with UV(S)S-A, indicating that UVSSA is the causative gene for this syndrome. The UVSSA protein forms a complex with USP7 (ref. 8), stabilizes ERCC6 and restores the hypophosphorylated form of RNA polymerase II after UV irradiation.
[Show abstract][Hide abstract] ABSTRACT: The xeroderma pigmentosum group F-cross-complementing rodent repair deficiency group 1 (XPF-ERCC1) complex is a structure-specific endonuclease involved in nucleotide excision repair (NER) and interstrand cross-link (ICL) repair. Patients with XPF mutations may suffer from two forms of xeroderma pigmentosum (XP): XP-F patients show mild photosensitivity and proneness to skin cancer but rarely show any neurological abnormalities, whereas XFE patients display symptoms of severe XP symptoms, growth retardation and accelerated aging. Xpf knockout mice display accelerated aging and die before weaning. These results suggest that the XPF-ERCC1 complex has additional functions besides NER and ICL repair and is essential for development and growth. In this study, we show a partial colocalization of XPF with mitotic spindles and Eg5. XPF knockdown in cells led to an increase in the frequency of abnormal nuclear morphology and mitosis. Similarly, the frequency of abnormal nuclei and mitosis was increased in XP-F and XFE cells. In addition, we showed that Eg5 enhances the action of XPF-ERCC1 nuclease activity. Taken together, these results suggest that the interaction between XPF and Eg5 plays a role in mitosis and DNA repair and offer new insights into the pathogenesis of XP-F and XFE.
Genes to Cells 03/2012; 17(3):173-85. DOI:10.1111/j.1365-2443.2012.01582.x · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BRCA1 is an important gene involved in susceptibility to breast and ovarian cancer and its product regulates the cellular response to DNA double-strand breaks. Here, we present evidence that BRCA1 also contributes to the transcription-coupled repair (TCR) of ultraviolet (UV) light-induced DNA damage. BRCA1 immediately accumulates at the sites of UV irradiation-mediated damage in cell nuclei in a manner that is fully dependent on both Cockayne syndrome B (CSB) protein and active transcription. Suppression of BRCA1 expression inhibits the TCR of UV lesions and increases the UV sensitivity of cells proficient in TCR. BRCA1 physically interacts with CSB protein. BRCA1 polyubiquitinates CSB and this polyubiquitination and subsequent degradation of CSB occur following UV irradiation, even in the absence of Cockayne syndrome A (CSA) protein. The depletion of BRCA1 expression increases the UV sensitivity of CSA-deficient cells. These results indicate that BRCA1 is involved in TCR and that a BRCA1-dependent polyubiquitination pathway for CSB exists alongside the CSA-dependent pathway to yield more efficient excision repair of lesions on the transcribed DNA strand.
Cancer Science 07/2011; 102(10):1840-7. DOI:10.1111/j.1349-7006.2011.02037.x · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early
stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition
to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding
regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer
and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays,
an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind
to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site
in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but
normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal
deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion.
Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.
[Show abstract][Hide abstract] ABSTRACT: Two UV-sensitive syndrome patients who have mild photosensitivity without detectable somatic abnormalities lack detectable Cockayne syndrome group B (CSB) protein because of a homozygous null mutation in the CSB gene. In contrast, mutant CSB proteins are produced in CS-B patients with the severe somatic abnormalities of Cockayne syndrome and photosensitivity. It is known that the piggyBac transposable element derived 3 is integrated within the CSB intron 5, and that CSB-piggyBac transposable element derived 3 fusion (CPFP) mRNA is produced by alternative splicing. We found that CPFP or truncated CSB protein derived from CPFP mRNA was stably produced in CS-B patients, and that wild-type CSB, CPFP, and truncated CSB protein interacted with DNA topoisomerase I. We also found that CPFP inhibited repair of a camptothecin-induced topoisomerase I-DNA covalent complex. The inhibition was suppressed by the presence of wild-type CSB, consistent with the autosomal recessive inheritance of Cockayne syndrome. These results suggested that reduced repair of a DNA topoisomerase I-DNA covalent complex because of truncated CSB proteins is involved in the pathogenesis of CS-B.
Genes to Cells 01/2011; 16(1):101-14. DOI:10.1111/j.1365-2443.2010.01467.x · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by sulfur-deficient brittle hair complicated with ichthyosis, physical and mental retardation, and proneness to infections. Approximately half of TTD patients exhibit cutaneous photosensitivity because of the defect of nucleotide excision repair. Three genes, XPB, XPD and TTDA, have been identified as causative genes of photosensitive TTD. These three genes are components of basal transcription factor IIH. Most TTD cases have been reported in Europe and North America. We report a severely affected Japanese TTD patient with XPD mutations. Interestingly, his father has ichthyotic skin. The alteration in the paternal allele was a nucleotide substitution leading to Arg-722 to Trp (R722W), as previously reported in TTD patients. The other alteration in the maternal allele was a novel 3-bp deletion at nucleotides 67-69, resulting in the deletion of Ser-23, which is located upstream of helicase motif I and is the closest to the N-terminal end of XPD in reported mutations. The expression study showed that the two alterations were causative mutations for TTD. In Asia, it is likely that there are TTD patients who have not been diagnosed.
Journal of Human Genetics 10/2010; 56(1):77-9. DOI:10.1038/jhg.2010.123 · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xeroderma pigmentosum group D (XPD) protein is one of the subunits of TFIIH that is required for nucleotide excision repair and transcription. We found a XPD protein complex containing MMS19 that was assumed to be a regulator of TFIIH. However, the MMS19-XPD complex did not contain any other subunits of TFIIH. Instead, it included FAM96B (now designated MIP18), Ciao1, and ANT2. MMS19, MIP18, and XPD localized to the mitotic spindle during mitosis. The siRNA-mediated knockdown of MMS19, MIP18, or XPD led to improper chromosome segregation and the accumulation of nuclei with abnormal shapes. In addition, the frequency of abnormal mitosis and nuclei was increased in XP-D and XP-D/CS patients' cells. These results indicate that the MMS19-XPD protein complex, now designated MMXD (MMS19-MIP18-XPD), is required for proper chromosome segregation, an abnormality of which could contribute to the pathogenesis in some cases of XP-D and XP-D/CS.
[Show abstract][Hide abstract] ABSTRACT: p53 suppresses the genomic instability provoked by genotoxic agents. Ultraviolet (UV) B induces skin cancers by producing
DNA damage and mutations in the skin genome, whereas the skin tissue responds to the UVB insult with cell cycle arrest and
apoptosis as well as damage exclusion by DNA repair. To address the p53 contribution to these skin responses in vivo, we analyzed the time course of DNA damage removal, apoptosis induction and hyperplasia in the skin after UVB irradiation
in p53-knockout mice. We also examined UVB-induced mutations in the skin. We found that p53 deficiency does not abolish the UVB-induced
apoptotic response in the epidermis but delays the process and the following hyperplasia 12–24 h. Regardless of the p53 genotype,
1 kJ/m2 UVB induced a total replacement of the epidermal layer by destroying the damaged epidermis by apoptosis and rebuilding a
new one through hyperplasia. We failed to detect a clear defect in removal of UVB-induced DNA photolesions from the genome
of the p53-deficient skin except for a delay in the epidermis, which seemed to result from the delay in the apoptotic response.
However, we found that p53 deficiency enhanced UVB-induced mutagenesis. Furthermore, in a genetic study using Xpa-knockout mice, we showed that the enhanced mutagenic response depends on the activity of nucleotide excision repair (NER),
which was also supported by the mutation spectrum observed in the UVB-exposed p53-knockout mice. These results indicate that p53 protects the skin genome from the UVB genotoxicity by facilitating NER, whereas
its contribution to the UVB-induced apoptosis is limited.
[Show abstract][Hide abstract] ABSTRACT: UV-damaged-DNA-binding protein (UV-DDB) is a heterodimer comprised of DDB1 and DDB2 and integrated in a complex that includes a ubiquitin ligase component, cullin 4A, and Roc1. Here we show that the ubiquitin ligase activity of the DDB2 complex is required for efficient global genome nucleotide excision repair (GG-NER) in chromatin. Mutant DDB2 proteins derived from xeroderma pigmentosum group E patients are not able to mediate ubiquitylation around damaged sites in chromatin. We also found that CSN, a negative regulator of cullin-based ubiquitin ligases, dissociates from the DDB2 complex when the complex binds to damaged DNA and that XPC and Ku oppositely regulate the ubiquitin ligase activity, especially around damaged sites. Furthermore, the DDB2 complex-mediated ubiquitylation plays a role in recruiting XPA to damaged sites. These findings shed some light on the early stages of GG-NER.
[Show abstract][Hide abstract] ABSTRACT: We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-alpha and cycloheximide. By affinity purification and mass spectrometry, RNA polymerase II-associated protein 3 (RPAP3) was identified as a Monad binding protein and may function with Monad as a novel modulator of apoptosis pathways. Here we report that Reptin, a highly conserved AAA + ATPase that is part of various chromatin-remodeling complexes, is also involved in the association of RPAP3 by immunoprecipitation and confocal microscopic analysis. Overexpression of RPAP3 induced HEK293 cells to death after UV-irradiation. Loss of RPAP3 by RNAi improved HeLa cell survival after UV-induced DNA damage and attenuated the phosphorylation of H2AX. Depletion of Reptin reduced cell survival and facilitated the phosphorylation on H2AX. These results suggest that RPAP3 modulates UV-induced DNA damage by regulating H2AX phosphorylation.
[Show abstract][Hide abstract] ABSTRACT: 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.
DNA Repair 10/2008; 7(12):1938-50. DOI:10.1016/j.dnarep.2008.08.003 · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleotide excision repair is a versatile repair pathway that counteracts the deleterious effects of various DNA lesions. In nucleotide excision repair, there is a transcription-coupled repair (TCR) pathway that focuses on DNA damage that blocks RNA polymerase IIo in transcription elongation. XAB2 (XPA-binding protein 2), containing tetratricopeptide repeats, has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A protein (XPA). Moreover, XAB2 has been shown to interact with Cockayne syndrome group A and B proteins (CSA and CSB) and RNA polymerase II, as well as XPA, and is involved in TCR and transcription. Here we purified XAB2 as a multimeric protein complex consisting of hAquarius, XAB2, hPRP19, CCDC16, hISY1, and PPIE, which are involved in pre-mRNA splicing. Knockdown of XAB2 with small interfering RNA in HeLa cells resulted in a hypersensitivity to killing by UV light and a decreased recovery of RNA synthesis after UV irradiation and regular RNA synthesis. Enhanced interaction of XAB2 with RNA polymerase IIo or XPA was observed in cells treated with DNA-damaging agents, indicating DNA damage-responsive activity of the XAB2 complex. These results indicated that the XAB2 complex is a multifunctional factor involved in pre-mRNA splicing, transcription, and TCR.