Research activity information

• Nov. 2016 第39回日本分子生物学会年会, 優秀ポスター賞, Palmドメインの欠失型変異体を用いたヒトDNAポリメラーゼ・イータの機能解析

  横井 雅幸

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

  Jun-ichi Akagi, Masayuki Yokoi, Yumi Miyake, Tsuyoshi Shirai, Tomohiro Baba, Young-Man Cho, Fumio Hanaoka, Kaoru Sugasawa, Shigenori Iwai, Kumiko Ogawa

  Elsevier BV, Jun. 2023, Journal of Biological Chemistry, 105002 - 105002

  [Refereed]

  Scientific journal


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

  Masayuki Kusakabe, Erina Kakumu, Fumika Kurihara, Kazuki Tsuchida, Takumi Maeda, Haruto Tada, Kanako Kusao, Akari Kato, Takeshi Yasuda, Tomonari Matsuda, Mitsuyoshi Nakao, Masayuki Yokoi, Wataru Sakai, Kaoru Sugasawa

  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.

  Elsevier BV, Apr. 2022, iScience, 25(4) (4), 104040 - 104040, English, International magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


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

  Wataru Sakai, Mayumi Yuasa-Sunagawa, Masayuki Kusakabe, Aiko Kishimoto, Takeshi Matsui, Yuki Kaneko, Jun-Ichi Akagi, Nicolas Huyghe, Masae Ikura, Tsuyoshi Ikura, Fumio Hanaoka, Masayuki Yokoi, Kaoru Sugasawa

  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.

  Nov. 2020, Scientific reports, 10(1) (1), 19704 - 19704, English, International magazine, Co-authored internationally

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


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

  Jun-Ichi Akagi, Keiji Hashimoto, Kenji Suzuki, Masayuki Yokoi, Niels de Wind, Shigenori Iwai, Haruo Ohmori, Masaaki Moriya, Fumio Hanaoka

  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.

  Dec. 2019, DNA repair, 87, 102771 - 102771, English, International magazine, Co-authored internationally

  [Refereed]


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

  Masayuki Kusakabe, Yuki Onishi, Haruto Tada, Fumika Kurihara, Kanako Kusao, Mari Furukawa, Shigenori Iwai, Masayuki Yokoi, Wataru Sakai, Kaoru Sugasawa

  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.

  BMC, 2019, Genes and environment : the official journal of the Japanese Environmental Mutagen Society, 41, 2 - 2, English, International magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• 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.

  Junichi Akagi, Masayuki Yokoi, Young-Man Cho, Takeshi Toyoda, Haruo Ohmori, Fumio Hanaoka, Kumiko Ogawa

  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.

  Jan. 2018, DNA repair, 61, 76 - 85, English, International magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


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

  Tomohumi Nakamura, Kouichi Murakami, Haruto Tada, Yoshihiko Uehara, Jumpei Nogami, Kazumitsu Maehara, Yasuyuki Ohkawa, Hisato Saitoh, Hideo Nishitani, Tetsuya Ono, Ryotaro Nishi, Masayuki Yokoi, Wataru Sakai, Kaoru Sugasawa

  Apr. 2017, GENES TO CELLS, 22(4) (4), 392 - 405, English

  [Refereed]

  Scientific journal


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

  Erina Kakumu, Seiya Nakanishi, Hiromi M. Shiratori, Akari Kato, Wataru Kobayashi, Shinichi Machida, Takeshi Yasuda, Naoko Adachi, Naoaki Saito, Tsuyoshi Ikura, Hitoshi Kurumizaka, Hiroshi Kimura, Masayuki Yokoi, Wataru Sakai, Kaoru Sugasawa

  Mar. 2017, GENES TO CELLS, 22(3) (3), 310 - 327, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


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

  Masayuki Yokoi, Fumio Hanaoka

  Lead, Elsevier B.V., Jan. 2017, Gene, 597, 1 - 9, English, International magazine

  [Refereed]

  Scientific journal


• UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι.

  Rie Kanao, Masayuki Yokoi, Tsuyoshi Ohkumo, Yasutaka Sakurai, Kantaro Dotsu, Shinobu Kura, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Chikahide Masutani, Fumio Hanaoka

  Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated.

  May 2015, DNA repair, 29, 139 - 46, English, International magazine

  [Refereed]

  Scientific journal


• Remarkable induction of UV-signature mutations at the 3'-cytosine of dipyrimidine sites except at 5'-TCG-3' in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice.

  Hironobu Ikehata, Yumin Chang, Masayuki Yokoi, Masayuki Yamamoto, Fumio Hanaoka

  The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh(-/-)) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh(-/-) epidermis and dermis than in the wild-type (Polh(+/+)) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh(-/-) mice were determined and compared with mutant sequences from irradiated Polh(+)(/)(+) mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C→T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5'-TCG-3' sequence context than at the other dipyrimidine contexts in the Polh(+/+) epidermis, whereas the mutations were induced remarkably and exclusively at the 3'-cytosine of almost all dipyrimidine contexts with no preference for 5'-TCG-3' in the Polh(-/-) epidermis. In addition, in Polh(-/-) mice, a small but remarkable fraction of G→T transversions was also observed exclusively at the 3'-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh(+/+) genotype.

  Oct. 2014, DNA repair, 22, 112 - 22, English, International magazine

  [Refereed]

  Scientific journal


• Identification of new scavengers for hydroxyl radicals and superoxide dismutase by utilising ultraviolet A photoreaction of 8-methoxypsoralen and a variety of mutants of Escherichia coli: implications on certain diseases of DNA repair deficiency.

  Shamim I Ahmad, Masayuki Yokoi, Fumio Hanaoka

  8-Methoxypsoralen+UVA (ultraviolet light of 320-400 nm) known as PUVA has been in use for a number of years for the treatment of psoriasis and vitiligo. The treatment possibly works on the basis of UVA photoactivated 8-methoxypsoralen binding to DNA forming both single strand and double strand type damage. We have used Escherichia coli as model system in studying PUVA induced DNA damage and repair. It has been known for some time that the photoactivated 8-methoxypsoralen, besides intercalating with DNA, generates at least two reactive oxygen species (ROS): hydroxyl radicals and superoxide anions, and also singlet oxygen. In this study it has been found that, in E. coli, malate dehydrogenase, succinate dehydrogenase and NADH:ubiquinone oxidoreductase can protect cells from PUVA killing presumably by scavenging these ROS. Possible mechanisms have been proposed for these enzymes as cell protectors. Studies also suggest the potential for the use of PUVA in the treatment of a large number of human diseases. This study also finds that, unlike 8-methoxypsoralen, trioxsalen (4,5',8-trimethylpsoralen, another derivative of psoralens) does not generate ROS by UVA photoactivation; and hence the mode of action of trioxsalen and PUVA overlaps only in the binding of these molecules to DNA in the presence of UVA.

  Nov. 2012, Journal of photochemistry and photobiology. B, Biology, 116, 30 - 6, English, International magazine, Co-authored internationally

  [Refereed]

  Scientific journal


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

  Wakana Ito, Masayuki Yokoi, Nobutaka Sakayoshi, Yasutaka Sakurai, Jun-Ichi Akagi, Hiroshi Mitani, Fumio Hanaoka

  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.

  Lead, Feb. 2012, Genes to cells : devoted to molecular & cellular mechanisms, 17(2) (2), 98 - 108, English, International magazine

  [Refereed]

  Scientific journal


• DNA polymerase eta is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation.

  Keiji Masuda, Rika Ouchida, Masayuki Yokoi, Fumio Hanaoka, Takachika Azuma, Ji-Yang Wang

  DNA polymerase eta (POLH) is required for the generation of A:T mutations during the somatic hypermutation of Ig genes in germinal center B cells. It remains unclear, however, whether POLH is a limiting factor for A:T mutations and how the absence of POLH might affect antibody affinity maturation. We found that the heterozygous Polh+/- mice exhibited a significant reduction in the frequency of A:T mutations in Ig genes, with each type of base substitutions at a level intermediate between the Polh+/+ and Polh(-/-) mice. These observations suggest that Polh is haplo-insufficient for the induction of A:T mutations in Ig genes. Intriguingly, there was also a reduction of C to T and G to A transitions in Polh+/- mice as compared with WT mice. Polh(-/-) mice produced decreased serum titers of high-affinity antibodies against a T-dependent antigen, which was associated with a significant reduction in the number of plasma cells secreting high-affinity antibodies. Analysis of the V region revealed that aa substitutions caused by A:T mutations were greatly reduced in Polh(-/-) mice. These results demonstrate that POLH is a limiting factor for A:T mutations and contributes to the efficient diversification of Ig genes and affinity maturation of antibodies.

  Oct. 2008, European journal of immunology, 38(10) (10), 2796 - 805, English, International magazine

  [Refereed]

  Scientific journal


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

  Stella A Martomo, Huseyin Saribasak, Masayuki Yokoi, Fumio Hanaoka, Patricia J Gearhart

  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.

  Sep. 2008, DNA repair, 7(9) (9), 1603 - 8, English, International magazine, Co-authored internationally

  [Refereed]

  Scientific journal


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

  Rika Ouchida, Akiko Ukai, Hiromi Mori, Kiyoko Kawamura, Martijn E T Dollé, Masatoshi Tagawa, Akemi Sakamoto, Takeshi Tokuhisa, Tadashi Yokosuka, Takashi Saito, Masayuki Yokoi, Fumio Hanaoka, Jan Vijg, Ji-Yang Wang

  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.

  Aug. 2008, DNA repair, 7(8) (8), 1392 - 8, English, International magazine

  [Refereed]

  Scientific journal


• DNA polymerases eta and theta function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes.

  Keiji Masuda, Rika Ouchida, Masaki Hikida, Tomohiro Kurosaki, Masayuki Yokoi, Chikahide Masutani, Mineaki Seki, Richard D Wood, Fumio Hanaoka, Jiyang O-Wang

  Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq(-/-)Polh(-/-) mice, however, did not show a further decrease of A/T mutations as compared with Polh(-/-) mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

  Jun. 2007, The Journal of biological chemistry, 282(24) (24), 17387 - 94, English, International magazine, Co-authored internationally

  [Refereed]

  Scientific journal


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

  Stella A Martomo, William W Yang, Alexandra Vaisman, Alex Maas, Masayuki Yokoi, Jan H Hoeijmakers, Fumio Hanaoka, Roger Woodgate, Patricia J Gearhart

  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.

  Mar. 2006, DNA repair, 5(3) (3), 392 - 8, English, International magazine, Co-authored internationally

  [Refereed]

  Scientific journal


• UV-B radiation induces epithelial tumors in mice lacking DNA polymerase η and mesenchymal tumors in mice deficient for DNA polymerase ι

  Ohkumo, T., Kondo, Y., Yokoi, M., Tsukamoto, T., Yamada, A., Sugimoto, T., Kanao, R., Higashi, Y., Kondoh, H., Tatematsu, M., Masutani, C., Hanaoka, F.

  Lead, 2006, Mol. Cell. Biol., 26, 7696 - 7706, English, International magazine

  [Refereed]

  Scientific journal


• Different mutation signatures in DNA polymerase eta- and MSH6-deficient mice suggest separate roles in antibody diversification.

  Stella A Martomo, William W Yang, Robert P Wersto, Tsuyoshi Ohkumo, Yuji Kondo, Masayuki Yokoi, Chikahide Masutani, Fumio Hanaoka, Patricia J Gearhart

  Hypermutation in immunoglobulin genes produces a high frequency of substitutions of all four bases, which are likely generated by low-fidelity DNA polymerases. Indeed, humans deficient for DNA polymerase (pol) eta have decreased substitutions of A.T base pairs in variable and switch regions. To study the role of pol eta in a genetically tractable system, we created mice lacking pol eta. B cells from Polh-/- mice produced normal amounts of IgG, indicating that pol eta does not affect class switch recombination. Similar to their human counterparts, variable and switch regions from Polh-/- mice had fewer substitutions of A.T base pairs and correspondingly more mutations of C.G base pairs, which firmly establishes a central role for pol eta in hypermutation. Notably, the location and types of substitutions differ markedly from those in Msh6-/- clones, which also have fewer A.T mutations. The data suggest that pol eta preferentially synthesizes a repair patch on the nontranscribed strand, whereas MSH6 functions to generate the patch.

  Jun. 2005, Proceedings of the National Academy of Sciences of the United States of America, 102(24) (24), 8656 - 61, English, International magazine, Co-authored internationally

  [Refereed]

  Scientific journal


• Studies of Schizosaccharomyces pombe TFIIE indicate conformational and functional changes in RNA polymerase II at transcription initiation

  Kazuhiro Hayashi, Tomomichi Watanabe, Aki Tanaka, Tadashi Furumuto, Chiaki Sato-Tsuchiya, Makoto Kimura, Masayuki Yokoi, Akira Ishihama, Fumio Hanaoka, Yoshiaki Ohkuma

  Mar. 2005, Genes to Cells, 10(3) (3), 207 - 224, English, International magazine

  [Refereed]

  Scientific journal


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

  Sano, S., Chan, K.S., Kira, M., Kataoka, K., Takagi, S., Tarutani, M., Itami, S., Kiguchi, K., Yokoi, M., Sugasawa, K., Mori, T., Hanaoka, F., Takeda, J., DiGiovanni, J.

  2005, Cancer Res., 65, 5720 - 5729, English, International magazine, Co-authored internationally

  [Refereed]


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

  Akio Uchida, Kaoru Sugasawa, Chikahide Masutani, Naoshi Dohmae, Marito Araki, Masayuki Yokoi, Yoshiaki Ohkuma, Fumio Hanaoka

  Jun. 2002, DNA Repair, 1(6) (6), 449 - 461, English, International magazine

  [Refereed]

  Scientific journal


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

  Fukumoto, Y., Hiyama, H., Yokoi, M., Nakaseko, Y., Yanagida, M., Hanaoka, F.

  2002, DNA Repair, 1, 833 - 845, English, International magazine

  [Refereed]

  Scientific journal


• E2F regulates growth-dependent transcription of genes encoding both catalytic and regulatory subunits of mouse primase

  NS Nishikawa, M Izumi, M Yokoi, H Miyazawa, F Hanaoka

  Jan. 2001, GENES TO CELLS, 6(1) (1), 57 - 70, English

  [Refereed]

  Scientific journal


• The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase

  M Izumi, K Yanagi, T Mizuno, M Yokoi, Y Kawasaki, KY Moon, J Hurwitz, F Yatagai, F Hanaoka

  Dec. 2000, NUCLEIC ACIDS RESEARCH, 28(23) (23), 4769 - 4777, English

  [Refereed]

  Scientific journal


• Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase a is controlled by E2F, an Ets-related transcription factor, and Sp1

  M Izumi, M Yokoi, NS Nishikawa, H Miyazawa, A Sugino, M Yamagishi, M Yamaguchi, A Matsukage, F Yatagai, F Hanaoka

  Jul. 2000, BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, 1492(2-3) (2-3), 341 - 352, English

  [Refereed]

  Scientific journal


• Cloning and characterization of the 5 '-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase alpha 68 kDa subunit gene (vol 28, 1525, 2000)

  NS Nishikawa, M Izumi, H Uchida, M Yokoi, H Miyazawa, F Hanaoka

  Jun. 2000, NUCLEIC ACIDS RESEARCH, 28(12) (12), U7 - U7, English

  [Refereed]

  Scientific journal


• Cloning and characterization of the 5 '-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase alpha 68 kDa subunit gene

  NS Nishikawa, M Izumi, H Uchida, M Yokoi, H Miyazawa, F Hanaoka

  Apr. 2000, NUCLEIC ACIDS RESEARCH, 28(7) (7), 1525 - 1534, English

  [Refereed]

  Scientific journal


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

  M Yokoi, C Masutani, T Maekawa, K Sugasawa, Y Ohkuma, F Hanaoka

  Lead, Mar. 2000, JOURNAL OF BIOLOGICAL CHEMISTRY, 275(13) (13), 9870 - 9875, English

  [Refereed]

  Scientific journal


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

  H Hiyama, M Yokoi, C Masutani, K Sugasawa, T Maekawa, K Tanaka, JHJ Hoeijmakers, F Hanaoka

  Sep. 1999, JOURNAL OF BIOLOGICAL CHEMISTRY, 274(39) (39), 28019 - 28025, English, Co-authored internationally

  [Refereed]

  Scientific journal


• The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta

  C Masutani, R Kusumoto, A Yamada, N Dohmae, M Yokoi, M Yuasa, M Araki, S Iwai, K Takio, F Hanaoka

  Jun. 1999, NATURE, 399(6737) (6737), 700 - 704, English

  [Refereed]

  Scientific journal


• The second-largest subunit of the mouse DNA polymerase α-primase complex facilitates both production and nuclear translocation of the catalytic subunit of DNA polymerase α

  Takeshi Mizuno, Nobutoshi Ito, Masayuki Yokoi, Akio Kobayashi, Katsuyuki Tamai, Hiroshi Miyazawa, Fumio Hanaoka

  American Society for Microbiology, 1998, Molecular and Cellular Biology, 18(6) (6), 3552 - 3562, English

  [Refereed]

  Scientific journal


• Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase or and its cell-cycle dependent expression

  M Yokoi, M Ito, M Izumi, H Miyazawa, H Nakai, F Hanaoka

  Nov. 1997, GENES TO CELLS, 2(11) (11), 695 - 709, English

  [Refereed]

  Scientific journal


• Identification of the nuclear localization signal of mouse DNA primase: Nuclear transport of p46 subunit is facilitated by interaction with p54 subunit

  T Mizuno, T Okamoto, M Yokoi, M Izumi, A Kobayashi, T Hachiya, K Tamai, T Inoue, F Hanaoka

  Nov. 1996, JOURNAL OF CELL SCIENCE, 109, 2627 - 2636, English

  [Refereed]

  Scientific journal

• 紫外線によるDNAの損傷とがん化のメカニズム (特集 紫外線が原因で起こる皮膚トラブルと化粧品開発)

  横井 雅幸, 菅澤 薫

  技術情報協会, Oct. 2016, Cosmetic stage, 11(1) (1), 9 - 15, Japanese


• 紫外線に誘発される突然変異においてDNAポリメラーゼ・イータとイオタが皮膚細胞と組織で果たす役割(Roles of DNA polymerases eta and iota in UV-induced mutagenesis of skin cells and tissues)

  櫻井 靖高, 横井 雅幸, 塚本 徹哉, 魏 民, 鰐渕 英機

  (一社)日本病理学会, Mar. 2015, 日本病理学会会誌, 104(1) (1), 393 - 393, Japanese


• 紫外線照射DNAの損傷乗り越え複製においてDNAポリメラーゼイータとイオタが果たす生理的役割(Physiological roles of DNA polymerases η and ι in translesion synthesis past UV-induced DNA damage)

  櫻井 靖高, 横井 雅幸, 塚本 徹哉, 小田 司, 魏 民, 山下 孝之, 鰐渕 英機, 立松 正衛, 村雲 芳樹, 花岡 文雄

  日本癌学会, Sep. 2014, 日本癌学会総会記事, 73回, P - 3076, English


• PolκのRev1に依存した活性が紫外線によるDNA損傷のTLSに関与する

  鈴木健司, 赤木純一, 赤木純一, 大橋英治, 横井雅幸, 大森治夫, 花岡文雄

  2012, 日本分子生物学会年会プログラム・要旨集(Web), 35th


• 立体構造解析から見えてきた損傷乗り越えDNA複製の分子メカニズム

  道津貫太郎, 横井雅幸, 花岡文雄

  放射線生物研究会, 2011, 放射線生物研究, 46(1) (1), 1 - 14, Japanese

  Introduction scientific journal


• UV誘発性突然変異およびDNA損傷トレランスにおけるDNAポリメラーゼηおよびιの役割(Roles of DNA polymerases η and ι in UV-induced mutagenesis and DNA damage tolerance)

  櫻井 靖高, 横井 雅幸, 大雲 剛志, 塚本 徹哉, 立松 正衛, 魏 民, 鰐渕 英機, 花岡 文雄

  (公社)日本生化学会, Dec. 2010, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 83回・33回, 2P - 0456, English


• UVB照射DNAの損傷乗り越え複製におけるDNAポリメラーゼイータとイオタの生体内での役割(Roles of DNA polymerases eta and iota in translesion synthesis past UVB-induced DNA lesions in vivo)

  櫻井 靖高, 横井 雅幸, 塚本 徹哉, 立松 正衛, 魏 民, 鰐渕 英機, 花岡 文雄

  日本癌学会, Aug. 2010, 日本癌学会総会記事, 69回, 523 - 524, English


• 哺乳類の多様なDNAポリメラーゼと疾患との関連

  横井雅幸, 花岡文雄

  Lead, 2010, G.I. Research, 18, 115 - 122, Japanese

  Introduction scientific journal


• Analysis of DNA damage response in Xpc-Polh-deficient mouse embryonic fibroblasts

  SAKURAI YASUTAKA, SUGIMOTO TAIKI, SAKAYOSHI NOBUTAKA, TANIGUCHI KOJI, TSUKAMOTO TETSUYA, TATEMATSU MASAE, YOKOI MASAYUKI, HANAOKA FUMIO

  31 Mar. 2007, Tissue culture research communications : the journal of experimental & applied cell culture research = 組織培養研究, 26(1) (1), 66 - 66, Japanese


• Identification and characterization of Ddb1 protein complex in Schizosaccharomyces pombe

  Yasunori Fukumoto, Fumi Amano, Naoshi Dohmae, Masayuki Yokoi, Fumio Hanaoka

  Jun. 2005, CELL STRUCTURE AND FUNCTION, 30, 41 - 41, English

  Summary international conference


• Functional analyses of Rhp23 protein in fission yeast nucleotide excision repair

  Masayuki Yokoi, Etsuko Tamura, Fumio Hanaoka

  May 2004, CELL STRUCTURE AND FUNCTION, 29, 63 - 63, English

  Summary international conference


• 皮膚癌と基本転写因子

  横井雅幸, 花岡文雄, 大熊芳明

  Lead, 現代医療社, 2000, 現代医療, 32(2) (2), 473 - 480, Japanese

  Introduction scientific journal


• General transcription tactors involved in cooperative regulation between repair of gene damages by the external reasons and transcription

  OHKUMA Yoshiaki, WATANABE Yoshinori, YAMAMOTO Seiji, FUJIMOTO Hiroyuki, YOKOI Masayuki, MAEKAWA Takafumi, HANAOKA Fumio

  01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 200 - 200, Japanese


• Analysis of mouse DNA polymerase α 54-kDa subunit gene promoter

  NISHIKAWA Naoko S., IZUMI Masako, YOKOI Masayuki, MIYAZAWA Hiroshi, HANAOKA Fumio

  01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 357 - 357, Japanese


• Studies on XPC functional domains

  UCHIDA Akio, MAEKAWA Takafumi, MASUTANI Chikahide, YOKOI Masayuki, SUGASAWA Kaoru, OHKUMA Yoshiaki, HANAOKA Fumio

  01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 370 - 370, Japanese


• The role of XPC-hHR23B complex in the repair complex in the repair complex formation

  YOKOI Masayuki, MASUTANI Chikahide, MAEKAWA Takafumi, OHKUMA Yoshiki, HANAIKA Fumio

  01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 370 - 370, Japanese


• Analysis of the interaction between hJR23A/B nad S5a

  HIYAMA Hideki, YOKOI Masayuki, MASUTANI Chikashide, SUGASAWA Kaoru, MAEKAWA Takafumi, TANAKA Keiji, HANAOKA Fumio

  01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 371 - 371, Japanese


• XPC-HHR23B蛋白質複合体とTFIIHの相互作用

  横井 雅幸, 前川 隆文, 益谷 央豪, 大熊 芳明, 花岡 文雄

  01 Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 778 - 778, Japanese


• ヌクレオチド除去修復に関与する蛋白質XPGとTFIIHの相互作用

  前川 隆文, 横井 雅幸, 益谷 央豪, 大熊 芳明, 花岡 文雄

  01 Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 778 - 778, Japanese


• DNA repair and cell cycle

  横井雅幸, 益谷央豪, 花岡文雄

  Lead, 1996, 蛋白質 核酸 酵素, 41, 1791 - 1798, Japanese

  Introduction scientific journal

• エッセンシャル遺伝学・ゲノム科学

  Hartl, Daniel L., 中村, 千春, 岡田, 清孝

  Joint translation, 12章, 化学同人, Jan. 2021, Japanese, ISBN: 9784759820485


• 紫外線によるDNAの損傷とがん化のメカニズム

  Yokoi,M, Sugasawa, K

  Joint work, 技術情報協会, Oct. 2016, Japanese

  General book


• 立体構造解析から見えてきた損傷乗り越えDNA複製の分子メカニズム

  道津 貫太郎, 横井 雅幸, 花岡 文雄

  Joint work, 放射線生物研究会, 2011, Japanese

  Scholarly book


• 哺乳類の多様なDNAポリメラーゼと疾患との関連

  横井 雅幸, 花岡文雄

  Joint work, 先端医学社, 2010, Japanese

  Scholarly book


• 皮膚癌と基本転写因子

  横井 雅幸, 花岡文雄, 大熊芳明

  Joint work, 現代医療社, 2000, Japanese

  Scholarly book


• DNA修復と細胞周期制御

  横井 雅幸, 益谷央豪, 花岡文雄

  Joint work, 共立出版, 1996, Japanese

  Scholarly book

• 脂肪族アルデヒドの代謝異常がゲノム安定性に及ぼす影響

  酒井恒, 䑺谷智也, 梶本武利, 岡田太郎, 篠原正和, 横井雅幸, 菅澤薫

  日本環境変異原ゲノム学会第53回大会, Dec. 2024, Japanese

  [Invited]

  Invited oral presentation


• ヌクレオチド除去修復のDNA損傷認識制御におけるヒストン修飾の役割

  綿田瑞希, 日下部将之, 前田拓海, 各務恵理菜, 横井雅幸, 酒井恒, 菅澤薫

  第47回日本分子生物学会年会, Nov. 2024, Japanese

  Poster presentation


• アルデヒド脱水素酵素ALDH3A2の欠損がゲノム安定性に及ぼす影響

  䑺谷智也, 岡田太郎, 梶本武利, 篠原正和, 横井雅幸, 菅澤薫, 酒井恒

  第47回日本分子生物学会年会, Nov. 2024, Japanese

  Poster presentation


• ヌクレオチド除去修復のDNA損傷認識を制御するクロマチン構造変換機構

  藤原叶枝, 槌田千輝, 日下部将之, 草尾佳那子, 八鍬聖, 横井雅幸, 酒井恒, 菅澤薫

  第47回日本分子生物学会年会, Nov. 2024, Japanese

  Poster presentation


• The involvement of chromatin remodeling factor SMACRAD1 in response to DNA double strand breaks

  Miou Takasu, Masayuki Kusanabe, Atsushi Shibata, Wataru Sakai, Kaoru Sugasawa, Masayuki Yokoi

  The12th 3R+3C International Symposium, Nov. 2024, English

  Poster presentation


• Impact of histone modifications on damage recognition process of global genome nucleotide excision repair

  Masayuki Kusakabe, Mizuki Watada, Takumi Maeda, Erina Kakumu, Kanae Fujiwara, Mizuki Otobe, Wataru Sakai, Masayuki Yokoi, Kaoru Sugasawa

  The12th 3R+3C International Symposium, Nov. 2024, English

  Poster presentation


• Contribution of translesion synthesis for mutagenesis via a novel food-induced formamidopyrimidine-derivative

  Jun-ichi Akagi, Masayuki Yokoi, Kohei Matsushita, Fumio Hanaoka, Kaoru Sugasawa, Shigenori Iwai, Kumiko Ogawa

  The12th 3R+3C International Symposium, Nov. 2024, English

  Poster presentation


• ヌクレオチド除去修復の損傷認識制御におけるヒストン修飾の役割

  日下部将之, 綿田瑞希, 前田拓海, 各務恵理菜, 藤原叶枝, 田口萌衣, 酒井恒, 横井雅幸, 菅澤薫

  日本放射線影響学会第67回大会, Sep. 2024, Japanese

  Oral presentation


• アクリルアミドの活性代謝物であるグリシドアミドのホルムアミドピリミジン誘導体による突然変異誘発機序

  赤木純一, 横井雅幸, 三宅ゆみ, 白井剛, 馬場智弘, 曺永晩, 花岡文雄, 菅澤薫, 岩井成憲, 小川久美子

  第83回日本癌学会学術総会, Sep. 2024, Japanese

  Oral presentation


• Contribution of translesion synthesis for mutagenesis via a novel food-induced formamidopyrimidine-derivative

  Jun-ichi Akagi, Masayuki Yokoi, Yumi Miyake, Tsuyoshi Shirai, Tomohiro Baba, Young-Man Cho, Fumio Hanaoka, Kaoru Sugasawa, Shigenori Iwai, Kumiko Ogawa

  7th DNA Polymerases Meeting, Aug. 2024, English

  Poster presentation


• グリシドアミド付加体のホルムアミドピリミジン誘導体はDNA複製阻害と突然変異を誘発する

  赤木純一, 横井雅幸, 三宅ゆみ, 白井剛, 馬場智弘, 曺永晩, 花岡文雄, 菅澤薫, 岩井成憲, 小川久美子

  第51回日本毒性学会学術年会, Jul. 2024, Japanese

  Poster presentation


• グリシドアミド付加体のホルムアミドピリミジン誘導体はDNA複製阻害と突然変異を誘発する

  赤木純一, 横井雅幸, 三宅ゆみ, 白井 剛, 馬場智弘, 曺 永晩, 花岡文雄, 菅澤 薫, 岩井成憲, 小川久美子

  日本薬学会年第144年会, Mar. 2024, Japanese

  Poster presentation


• 損傷クロマチン基質を用いたヌクレオチド除去修復制御機構の生化学的解析

  古園英嗣, 多田遥人, 日下部将之, 松本翔太, 横井雅幸, 酒井 恒, 鯨井智也, 胡桃坂仁志, 菅澤 薫

  第41回染色体ワークショップ・第22回核ダイナミクス研究会, Jan. 2024, Japanese

  Poster presentation


• ヌクレオチド除去修復の損傷監視制御におけるヒストン修飾の役割

  日下部将之, 綿田瑞希, 前田拓海, 各務恵理菜, 藤原叶枝, 乙部瑞貴, 酒井 恒, 横井雅幸, 菅澤 薫

  第41回染色体ワークショップ・第22回核ダイナミクス研究会, Jan. 2024, Japanese

  Oral presentation


• ヌクレオチド除去修復のDNA損傷認識制御におけるヒストンH3K9メチル化修飾の役割

  綿田瑞希, 日下部将之, 前田拓海, 各務恵理菜, 横井雅幸, 酒井 恒, 菅澤 薫

  第46回日本分子生物学会年会, Dec. 2023, Japanese

  Poster presentation


• 紫外線誘発DNA損傷に対する細胞応答におけるクロマチンリモデリング因⼦SMARCAD1の関与

  高須美央, 坂口友美, 酒井麻衣, 鹿谷有由希, 福永 匠, 酒井 恒, 日下部将之, 菅澤 薫, 花岡文雄, 横井雅幸

  第46回日本分子生物学会年会, Dec. 2023, Japanese

  Poster presentation


• DNAポリメラーゼ‧イータの発現調節における脱ユビキチン化酵素USP11の関与

  中森晴渚, 仲野由佳梨, 案済 萌, 菅野新一郎, 安井 明, 酒井 恒, 菅澤 薫, 花岡文雄, 横井雅幸

  第46回日本分子生物学会年会, Dec. 2023, Japanese

  Poster presentation


• ゲノム全体を対象としたヌクレオチド除去修復の損傷認識を制御するクロマチン動態

  日下部将之, 前田拓海, 綿田瑞希, 藤原叶枝, 各務恵理菜, 乙部瑞貴, 酒井 恒, 横井雅幸, 菅澤 薫

  第46回日本分子生物学会年会, Dec. 2023, Japanese

  Oral presentation


• ⾷品汚染物質アクリルアミドの活性代謝物により⽣じるホルムアミドピリミジン誘導体の突然変異誘発機構

  赤木純一, 横井雅幸, 三宅ゆみ, 白井 剛, 馬場智弘, 曺 永晩, 花岡文雄, 菅澤 薫, 岩井成憲, 小川久美子

  第46回日本分子生物学会年会, Dec. 2023, Japanese

  Oral presentation


• ヒストン翻訳後修飾を介したXPCタンパク質の核内局在制御

  乙部瑞貴, 日下部将之, 各務恵理菜, 槌田千輝, 塩月陽人, 酒井 恒, 横井雅幸, 菅澤 薫

  第46回日本分子生物学会年会, Dec. 2023, Japanese

  Poster presentation


• ヌクレオチド除去修復のDNA損傷認識を制御するクロマチン構造変換機構

  藤原叶枝, 槌田千輝, 日下部将之, 草尾佳那子, 八鍬 聖, 酒井 恒, 横井雅幸, 菅澤 薫

  第46回日本分子生物学会年会, Dec. 2023, Japanese

  Poster presentation


• 脂肪族アルデヒド脱水素酵素の欠損により生じる細胞毒性の解析

  䑺谷智也, 大槻侑恵, 後藤元成, 笹野真唯子, 松田 俊, 松田知成, 梶本武利, 岡田太郎, 横井雅幸, 菅澤 薫, 酒井 恒

  日本環境変異原ゲノム学会第52回大会, Nov. 2023, Japanese

  Poster presentation


• 脂肪族アルデヒド脱水素酵素のゲノム安定性維持における影響

  酒井 恒, 䑺谷智也, 大槻侑恵, 後藤元成, 笹野真唯子, 松田 俊, 松田知成, 梶本武利, 岡田太郎, 横井雅幸, 菅澤 薫

  日本環境変異原ゲノム学会第52回大会, Nov. 2023, Japanese

  Oral presentation


• 脂質代謝制御におけるファンコニ貧血タンパク質の機能解析

  酒井 恒, 䑺谷智也, 大槻侑恵, 後藤元成, 乾 愛実, 松田 俊, 松田知成, 横井雅幸, 菅澤 薫

  第45回日本分子生物学会年会, Nov. 2022, Japanese

  Poster presentation


• ヌクレオチド除去修復におけるDNA損傷認識を制御するクロマチン構造変換因子複合体

  槌田千輝, 日下部将之, 各務恵理菜, 栗原文佳, 草尾佳那子, 前田拓海, 横井雅幸, 酒井 恒, 菅澤 薫

  第45回日本分子生物学会年会, Nov. 2022, Japanese

  Poster presentation


• 脂質代謝制御におけるファンコニ貧血タンパク質の機能解析

  酒井 恒, 䑺谷智也, 大槻侑恵, 後藤元成, 乾 愛実, 松田 俊, 松田知成, 横井雅幸, 菅澤 薫

  第45回日本分子生物学会年会, Nov. 2022, Japanese

  Oral presentation


• Ring-Opened N7-deoxyguanosine adduct of glycidamide induces DNA replication inhibition and mutagenesis

  Jun-ichi Akagi, Masayuki Yokoi, Young-Man Cho, Fumio Hanaoka, KaoruSugasawa, Shigenori Iwai, Kumiko Ogawa

  第49回国際核酸化学シンポジウム, Nov. 2022, English

  Poster presentation


• ヌクレオチド除去修復における紫外線誘発DNA損傷の効率的な認識に寄与するヒストン修飾

  日下部将之, 前田拓海, 各務恵理菜, 綿田瑞希, 酒井 恒, 横井雅幸, 菅澤 薫

  日本放射線影響学会第65回大会, Sep. 2022, Japanese

  Oral presentation


• 損傷乗り越え型DNAポリメラーゼPolκとREV1はグリシドアミドN7位デオキシグアノシン付加体による点突然変異に寄与する

  赤木純一, 横井雅幸, Young-Man Cho, 岩井成憲, 花岡文雄, 菅澤薫, 小川久美子

  日本薬学会第142年会, Mar. 2022, Japanese, Domestic conference

  Poster presentation


• DNAポリメラーゼの活性を阻害する低分子化合物の解析

  佐藤正康, 横井雅幸

  神戸大学研究基盤センター若手研究フロンティア研究会2021, Dec. 2021, Japanese, Domestic conference

  Poster presentation


• ヒストン脱アセチル化はヌクレオチド除去修復におけるDNA損傷認識を制御する

  槌田千輝, 日下部将之, 各務恵理菜, 栗原文佳, 多田遥人, 前田拓海, 横井雅幸, 酒井恒, 菅澤薫

  第44回日本分子生物学会年会, Dec. 2021, Japanese, Domestic conference

  Poster presentation


• 色素性乾皮症C群タンパク質によるDNA損傷認識を制御するクロマチン動態

  前田拓海, 日下部将之, 横井雅幸, 酒井恒, 菅澤薫

  第44回日本分子生物学会年会, Dec. 2021, Japanese, Domestic conference

  Poster presentation


• ファンコニ貧血タンパク質の脂質代謝における機能解析

  䑺谷智也, 大槻侑恵, 後藤元成, 乾愛実, 横井雅幸, 菅澤薫, 酒井恒

  第44回日本分子生物学会年会, Dec. 2021, Japanese, Domestic conference

  Poster presentation


• 脱ユビキチン化酵素による損傷乗り越え合成の制御機構の解析

  仲野由佳梨, 案済萌, 菅野新一郎, 安井明, 酒井恒, 菅澤薫, 花岡文雄, 横井雅幸

  第44回日本分子生物学会年会, Dec. 2021, Japanese, Domestic conference

  Poster presentation


• グリシドアミドN7位デオキシグアノシン付加体による点突然変異に寄与する損傷乗り越え型DNAポリメラーゼの解析

  赤木純一, 横井雅幸, Young-Man Cho, 岩井成憲, 花岡文雄, 菅澤薫, 小川久美子

  第44回日本分子生物学会年会, Dec. 2021, Japanese, Domestic conference

  Poster presentation


• ヌクレオチド除去修復におけるDNA損傷認識を制御するクロマチンダイナミクス

  日下部将之, 各務恵理菜, 栗原文佳, 槌田千輝, 多田遥人, 横井雅幸, 酒井恒, 菅澤薫

  第94回日本生化学会大会, Nov. 2021, Japanese, Domestic conference

  Oral presentation


• The N7-glycidamide adduct of 2’-deoxyguanosine on the template strand induces DNA replication inhibition and mutagenesis in human cells

  Jun-ichi Akagi, Masayuki Yokoi, Young-Man Cho, Shigenori Iwai, Fumio Hanaoka, Kaoru Sugasawa, Kumiko Ogawa

  第47回日本毒性学会学術年会, Jun. 2020, English

  Poster presentation


• アクリルアミドの活性代謝物であるグリシルアミドのデオキシグアノシンN7位付加体はDNA複製阻害と点突然変異を誘発する

  赤木純一, 横井雅幸, Young-Man Cho, 岩井成憲, 花岡文雄, 菅澤 薫, 小川久美子

  日本薬学会第140年会, Japanese, Kyoto, Japan, Domestic conference

  Poster presentation


• N7-グリシドアミドdG付加体により誘発されるDNA複製阻害と変異原性の解析

  赤木純一, 横井雅幸, Young-Man Cho, 岩井成憲, 花岡文雄, 菅澤 薫, 小川久美子

  第42回日本分子生物学会年会, English, Fukuoka, Japan, Domestic conference

  Poster presentation


• RNAを介したヌクレオチド除去修復制御機構の解析

  古川真理, 日下部将之, 酒井 恒, 横井雅幸, 菅澤 薫

  第42回日本分子生物学会年会, English, Fukuoka, Japan, Domestic conference

  Poster presentation


• 脂質代謝に応答したファンコニ貧血タンパク質のダイナミクス

  酒井 恒, 乾 愛実, 大槻侑恵, 後藤元成, 横井雅幸, 菅澤 薫

  第42回日本分子生物学会年会, English, Fukuoka, Japan, Domestic conference

  Poster presentation


• ファンコニ貧血タンパク質FANCD2と脂質代謝関連因子の機能的連関の解析

  大槻侑恵, 後藤元成, 乾 愛実, 松田 俊, 松田知成, 横井雅幸, 菅澤 薫, 酒井 恒

  第42回日本分子生物学会年会, English, Fukuoka, Japan, Domestic conference

  Poster presentation


• DNAポリメラーゼ・イータと脱ユビキチン化酵素の相互作用の解析

  案済 萌, 西村 潤, 菅野新一郎, 安井 明, 酒井 恒, 菅澤 薫, 花岡文雄, 横井雅幸

  第42回日本分子生物学会年会, English, Fukuoka, Japan, Domestic conference

  Poster presentation


• TLSポリメラーゼの損傷部位への導入過程を生細胞イメージングにより解析する試み

  福永 匠, 尾崎未羽, 佐藤正康, 酒井 恒, 菅澤 薫, 花岡文雄, 横井雅幸

  第42回日本分子生物学会年会, Japanese, FUKUOKA, Japan, Domestic conference

  Poster presentation


• XPCタンパク質によるDNA損傷認識の制御とヒストン修飾の役割

  栗原文佳, 日下部将之, 各務恵理菜, 草尾佳那子, 前田拓海, 横井雅幸, 酒井 恒, 菅澤 薫

  第42回日本分子生物学会年会, Japanese, FUKUOKA, Japan, Domestic conference

  Poster presentation


• ヌクレオチド除去修復の損傷認識を制御するクロマチン構造変換因子の探索

  草尾佳那子, 日下部将之, 各務恵理菜, 栗原文佳, 前田拓海, 横井雅幸, 酒井 恒, 菅澤 薫

  第42回日本分子生物学会年会, Japanese, FUKUOKA, Japan, Domestic conference

  Poster presentation


• Chromatin dynamics regulating DNA lesion recognition in nucleotide excision repair

  Masayuki Kusakabe, Fumika Kurihara, Kanako Kusao, Masayuki Yokoi, Wataru Sakai, Kaoru Sugasawa

  6th Asian Congress on Environmental Mutagens (ACEM) and 48th Annual Meeting of the Japanese Environmental Mutagen Society (JEMS), Nov. 2019, English, Tokyo, Japan, International conference

  [Invited]

  Invited oral presentation


• Dynamics of FANCD2 in response to lipid metabolism

  Yukie Otsuki, Motonari Goto, Megumi Inui, Masayuki Yokoi, Kaoru Sugasawa, Wataru Sakai

  2019 Fanconi Anemia Scientific Symposium, Sep. 2019, English, CHICAGO, United States, International conference

  Poster presentation


• XPCタンパク質によるDNA損傷認識の制御におけるヒストン修飾の役割

  KURIHARA FUMIKA, KUSAKABE MASAYUKI, KATO AKARI, KAKUMU ERINA, NAKANISHI SEIYA, KUSAO KANAKO, OKAWA YASUYUKI, YOKOI MASAYUKI, SAKAI WATARU, SUGASAWA KAORU

  第41回日本分子生物学会年会, Nov. 2018, Japanese, 横浜, Domestic conference

  Poster presentation


• Translesion synthesis-associated chromatin remodeling factors contribute the function of human DNA polymerase η in cisplatin-treated cells.

  Masayuki Yokoi, Yuuki Shikaya, Kaoru Sugasawa, Fumio Hanaoka

  The 11th 3R&3C Symposium, Nov. 2018, English, Kanazawa Japan, International conference

  Oral presentation


• N7-glycidamide-dG adduct inhibits DNA replication in mammalian cells

  AKAGI JUNICHI, YOKOI MASAYUKI, CHO YOUNG-MAN, YAMAMOTO J, MIZUTA YASUKO, IWAI SHIGENORI, HANAOKA FUMIO, OGAWA KUMIKO

  第41回日本分子生物学会年会, Nov. 2018, Japanese, 横浜, Domestic conference

  Poster presentation


• DNAポリメラーゼ・イータと脱ユビキチン化酵素の相互作用の解析

  ANZAI MOE, NISHIMURA JUN, KANNO SHINICHIRO, YASUI AKIRA, SUGASAWA KAORU, HANAOKA FUMIO, YOKOI MASAYUKI

  第41回日本分子生物学会年会, Nov. 2018, English, 横浜, Domestic conference

  Poster presentation


• Benzo[a]pyrene-induced tumorigenesis in Polκ-knockout mice

  AKAGI JUNICHI, CHO Young-Man, TOYODA TAKESHI, YOKOI MASAYUKI, HANAOKA FUMIO, OHMORI HARUO, OGAWA KUMIKO

  The 11th 3R+3C Symposium, Nov. 2018, English, 金沢, International conference

  Poster presentation


• クロマチン構造を介したヌクレオチド除去修復の高次制御機構

  日下部将之, 各務恵理菜, 加藤安佳梨, 栗原文佳, 草尾佳那子, 横井雅幸, 酒井 恒, SUGASAWA KAORU

  第91回生化学会大会, Sep. 2018, Japanese, 京都, Domestic conference

  Oral presentation


• Effect of Polκ deficiency on benzo[a]pyrene-induced tumorigenesis

  AGAGI JUNICHI, YOUNG-MAN CHO, TOYODA TAKESHI, YOKOI MASAYUKI, HANAOKA FUMIO, OGAWA KUMIKO

  The 5th DNA polymerase meeting, Sep. 2018, English, Leiden, The Netherlands, International conference

  Poster presentation


• DNA損傷による複製の停止を回避する機構とその制御

  YOKOI Masayuki

  神戸大学第6回バイオシグナル研究会, Mar. 2018, Japanese, 神戸大学百年記念館, Domestic conference

  Oral presentation


• Analysis of contribution of Polκ to benzo[a]pyrene-induced tumorigenesis

  AKAGI JUNICH, YOUNG-MAN CHO, TOYODA TAKESHI, MIZUTA YASUKO, YOKOI MASAYUKI, HANAOKA FUMIO, OMORI HARUO, OGAWA KUMIKO

  日本薬学会第138回, Mar. 2018, Japanese, 金沢, Domestic conference

  Poster presentation


• Contribution of Polκ to tumorigenesis in the forestomach of benzo[a]pyrene-fed mice

  AKAGI JUNICHI, YOUNG-MAN CHO, TOYODA TAKESHI, MIZUTA YASUKO, YOKOI MASAYUKI, HANAOKA FUMIO, OHMORI HARUO, OGAWA KUMIKO

  2017年度生命科学系学会合同年次大会, Dec. 2017, Japanese, 神戸国際会議場, Domestic conference

  Poster presentation


• シスプラチン誘発DNA鎖内架橋の乗り越え合成に関わるヒストン修飾とクロマチン構造変換因子の探索

  YOKOI MASAYUKI, SHIKAYA YUUKI, SUGASAWA KAORU, 花岡 文雄

  第24回 DNA複製・組換え・修復ワークショップ, Nov. 2017, Japanese, 長良川国際会議場, Domestic conference

  Poster presentation


• 損傷乗り越え型DNAポリメラーゼ イータ・イオタ・カッパ三重欠損細胞を用いた新規遺伝毒性試験法の研究

  AKAGI JUNICHI, YOKOI MASAYUKI, YONG-MAN CHO, TOYODA TAKESHI, OHMORI HARUO, HANAOKA FUMIO, OGAWA KUMIKO

  日本薬学会レギュラトリーサイエンス部会, Sep. 2017, Japanese, 慶應義塾大学 薬学部, Domestic conference

  Poster presentation


• Chromatin remodeling factors contribute to the function of human DNA polymerase η in the cells treated with cisplatin

  YOKOI MASAYUKI, SUGASAWA KAORU, HANAOKA FUMIO

  第76回 日本癌学会, Sep. 2017, Japanese, パシフィコ横浜, Domestic conference

  Poster presentation


• Functional analysis of human DNA polymerase eta harboring mutations in the palm domain

  Yokoi,M, Mishima,K, Noda,C., Nishimura, Yang,W, Hanaoka,F

  第39回 日本分子生物学会年会, Dec. 2016, Japanese, 日本分子生物学会, 横浜, Domestic conference

  Poster presentation


• Triple knockout mouse fibroblast cells defective for DNA polymerases eta, iota, and kappa exhibit hypersensitivity to various genotoxic mechanisms and have potential for screening of chemical genotoxicity

  Akagi,J, Yokoi,M, Cho,Y.-M, Toyota,T, Ohmori,H, Hanaoka,F, Ogawa,K

  The 10th 3R Symposium, Nov. 2016, English, 10th 3R organizing committee, 松江, International conference

  Poster presentation


• Translesion DNA polymerase η, ι, and κ triple knockout cells exhibit hypersensitivity to unmetabolized benzo[a]pyrene

  Akagi, J, Yokoi, M, Cho, Y.-M, Toyota, T, Ohmori, H, Hanaoka, F, Ogawa, K

  第39回 日本分子生物学会, Nov. 2016, Japanese, 横浜, Domestic conference

  Poster presentation


• Structure and function relations of human DNA polymerase eta: mutations of the finger or little finger domains alter lesion bypass efficiencies

  Yokoi,M, Onaka,S, Mishima,K, Yang,W, Hanaoka,F

  The 10th 3R Symposium, Nov. 2016, English, 10th 3R organizing committee, 松江, International conference

  Poster presentation


• ヒトPoletaの構造的特徴と損傷乗り越え活性の連関

  Yokoi,M

  国立遺伝学研究所研究集会「生物ゲノム安定維持の分子機構」, Oct. 2016, Japanese, 国立遺伝学研究所, 三島, Domestic conference

  Oral presentation


• Mutant analyses of human DNA polymerase eta based on high-resolution crystal structures of the protein-DNA-dNTP complex

  Hanaoka,F, Yokoi,M, Masutani,C, Yang,W

  DNA polymerase Meeting: from Molecular Function to Human Diseases, Oct. 2016, English, Biarritz,France, International conference

  [Invited]

  Invited oral presentation


• Deficiency of Pols eta/iota/kappa exhibit different sensitivities to various chemicals and are useful for screening of genotoxity

  Akagi,J, Yokoi,M, Cho,Y.-M, Toyota,T, Ohmori,H, Hanaoka,F, Ogawa,K

  第75回 日本癌学会学術総会, Oct. 2016, Japanese, 日本癌学会, 横浜, Domestic conference

  Poster presentation


• Cancer and translesion synthesis polymerases: with special reference on UV-induced mutagenesis

  Sakurai,Y, Yokoi,M, Hanaoka,F

  Cold Spring Harbor Asia Conference: DNA Metabolism, Genomic Stability & Diseases, Jun. 2016, English, Cold Spring Habor Laboratory, Suzhou,China, International conference

  [Invited]

  Invited oral presentation


• Analysis of the interaction between hJR23A/B nad S5a

  HIYAMA Hideki, YOKOI Masayuki, MASUTANI Chikashide, SUGASAWA Kaoru, MAEKAWA Takafumi, TANAKA Keiji, HANAOKA Fumio

  日本分子生物学会年会プログラム・講演要旨集, Dec. 1998, Japanese

• 日本遺伝学会

  Jun. 2023 - Present


• The Japanese Cancer Association

  May 2009 - Present


• The Molecular Biology Society of Japan

  Nov. 1992 - Present

• TLSポリメラーゼの安定性を介した脱ユビキチン化酵素のゲノム安定性への寄与

  横井 雅幸, 安井 明

  東北大学, 東北大学加齢医学研究所共同利用・共同研究, 神戸大学バイオシグナル総合研究センター, Apr. 2023 - Mar. 2024, Principal investigator


• ゲノム複製の停止と再開に関連したTLS機構とクロマチン構造変換のクロストーク

  横井 雅幸

  日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 神戸大学, 01 Apr. 2020 - 31 Mar. 2023

  【ヒトTLS因子の翻訳後修飾に関わる因子の探索と解析】ヒト損傷乗り越えDNA合成（TLS）関連因子のユビキチン化・脱ユビキチン化は、TLS機構の制御において重要である。TLSで主要な働きを担うDNAポリメラーゼ・イータ（POLH）のユビキチン化を担うE3ユビキチンリガーゼ（E3）を探索するため、288種類のRINGフィンガー型E3との試験管内相互作用の強さをもとに、上位１０種類の候補因子を選定した。発現抑制による内在性POLHの安定性を紫外線照射の有無で比較したところ、紫外線未照射時の分解への関与が示唆されたE3を４種類、照射時の分解への関与が示唆されたE3を２種類、新規に同定した。 【TLS反応に関与するクロマチン構造変換因子の解析】POLHのDNA損傷部位へのリクルートに関わる可能性が示唆されたクロマチン構造変換因子のうち、SMARCAD1を発現抑制したS期同調細胞の核に局所紫外線を照射してPOLHの挙動を調べた結果、効率的なPOLHのリクルートが有意に低下した。同様なPOLHのリクルートの有意な低下は、CHD9の発現抑制でも確認された。 【TLS反応を特異的に阻害する化合物の探索】１２種類のDNAポリメラーゼのDNA合成活性に対する特異性解析の結果をもとに、POLHに対する特異性が比較的高いと判断した４種類の低分子化合物について基質DNAとの親和性を解析した。その結果、３種類がDNA結合を阻害し、１種類はDNA結合に影響しなかった。DNA結合を阻害した３種類について酵素反応速度論的解析を行なった結果、その阻害様式として、２種類は競合的作用し、１種類は非競合的に作用することを明らかにした。さらに、DNA結合に影響しなかった１種類について、酵素反応速度論的解析によりヌクレオチドの取り込みから重合までの過程を調べた結果、その阻害様式が競合的であることを示した。


• TLSポリメラーゼの安定性調節機構と紫外線誘発皮膚発がんに関する研究

  横井 雅幸, 安井 明

  東北大学, 東北大学加齢医学研究所共同利用・共同研究, 神戸大学バイオシグナル総合研究センター, Apr. 2020 - Mar. 2023, Principal investigator


• Molecular mechanism of acrylamide-induced mutagenesis via translesion synthesis

  Akagi Jun-ichi

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), National Institute of Health Sciences, Apr. 2019 - Mar. 2022, Coinvestigator

  We generated knockout cells for Polη, Polι, Polκ, and REV1 to identify the DNA polymerases that contribute to acrylamide-induced mutations in human cells. The site-specific mutation assay using the stable analogue of N7-dG adduct of glycidamide (GA7dG), the active metabolite of acrylamide revealed that Polκ and REV1 partially contribute to the GA7dG-induced mutations via translesion DNA synthesis, which induce G:C > A:T and G:C > C:G point mutations at the lesion.


• グアニン四重鎖構造に関連する複製ストレスにおける損傷乗り越えDNA 合成ポリメラー ゼの役割

  横井 雅幸

  群馬大学, 群馬大学生体調節研究所内分泌・代謝学共同研究拠点共同研究, 神戸大学バイオシグナル総合研究センター, Apr. 2020 - Mar. 2021, Principal investigator


• 上皮系皮膚がんと間葉系皮膚繊維肉腫の紫外線による発症を異なるDNAポリメラーゼが抑制するメカニズムの研究

  横井 雅幸, 安井 明

  東北大学, 東北大学加齢医学研究所共同利用・共同研究, 神戸大学バイオシグナル総合研究センター, Apr. 2017 - Mar. 2020, Principal investigator


• Studies on the molecular mechanisms to regulate the resumption of stalled replication by translesion synthesis at DNA damage sites

  YOKOI MASAYUKI

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2017 - Mar. 2020, Principal investigator

  The aim of this research is to elucidate the fundamental mechanism(s) which is important for the resumption of stalled DNA replication by translesion synthesis (TLS) at DNA damage sites. DNA replication arrest at DNA lesion induces post-translational modifications (PTM) of TLS factor(s) such as replication accessary protein PCNA and/or TLS polymerase(s) on chromatin. In order to clarify how TLS governs and controls the resumption of chromosomal replication stalled at the DNA damage on chromatin, the effects of PTM on the function of TLS factors and its relation on chromatin organization were studied. As a result of analysis, new discoveries regarding the stability control of TLS polymerase, the relation of TLS and chromatin organization, and small molecules which inhibit TLS polymerase were obtained.


• Analysis of the molecular mechanisms of acrylamide-induced mutagenesis by using intracellular translesion synthesis assay

  Akagi Jun-ichi, Iwai Shigenori, Hanaoka Fumio, Yokoi Masayuki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), National Institute of Health Sciences, Apr. 2016 - Mar. 2019, Coinvestigator not use grants

  Acrylamide (AA) is a genotoxic carcinogen that forms by cooking foods at high temperature. In this study, we performed translesion DNA synthesis assay by using DNA template carrying GA7FdG, a stable analogue of dG adduct of glycidamide, the active metabolite of AA, to examine the molecular mechanism of AA-induced genotoxicity. We found that DNA synthesis by all DNA polymerases tested almost completely stalled at GA7FdG on the template strand. Moreover, replication efficiency of a shuttle vector carrying GA7FdG is significantly reduced in human cells. Base substitution mutations were observed in progeny of the GA7FdG strand. These results indicated that DNA replication arrest and mutagenesis by GA adducts directly contributes to AA-induced genotoxicity.


• Functional analyses of translesion synthesis DNA polymerase Poleta based on its structural features

  YOKOI MASAYUKI

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Apr. 2014 - Mar. 2018, Principal investigator

  Human Poleta is a major translesion synthesis polymerase involved in DNA damage tolerance against UV- or cisplatin-induced DNA lesions. To elucidate its physiological function and regulatory mechanism in cellular DNA damage tolerance, I focused on unique structural features of human Poleta identified from the comparison of its crystal structure against other eukaryotic TLS polymerases. As a result, unique amino acid sequences or amino acid residues of human Poleta were deleted or substituted by other amino acid to investigate their role. Biochemical and cellular analyses using these mutant proteins revealed that some of them were important for the translesion synthesis activity or regulation of human Poleta. It was notable, in this research, that several novel proteins and chemical compounds were identified to interact with or regulate human Poleta. These findings will open new phase of research on DNA damage tolerance associated with human Poleta.


• 突然変異を決定するヒトTLSポリメラーゼの結合タンパク質の同定と発がん・老化・免疫機能との関与

  横井 雅幸, 花岡 文雄, 安井 明

  東北大学, 年度東北大学加齢医学研究所 共同利用・共同研究公募要項, 学習院大学, Apr. 2015 - Mar. 2016, Principal investigator


• 細胞老化と発がんにおける複製ストレス・シグナルとクロマチン動態の解明

  横井 雅幸, 花岡 文雄

  群馬大学, 群馬大学生体調節研究所内分泌・代謝学共同研究拠点共同研究, 学習院大学, Apr. 2014 - Mar. 2015, Principal investigator


• Generality of translesion synthesis coupling replication and repair

  HANAOKA FUMIO, MASUTANI Chikahide, YOKOI Masayuki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Gakushuin University, Apr. 2010 - Mar. 2015, Coinvestigator not use grants

  In order to examine the generality of translesion synthesis (TLS), which coordinates replication and repair, we analyzed the structure of co-crystals of human DNA polymerase eta (Pol eta) and CPD-containing DNA with X-ray diffraction. We found that human Pol eta acts like a molecular splint to stabilize damaged DNA in a normal B-form conformation. On the other hand, the crystal structure of human Pol eta and cisplatin-containing DNA revealed that Pol eta could not act like a molecular splint. Mono- or polyubiquitination of PCNA controls the pathway choice of DNA damage tolerance. We demonstrated that PCNA is poly-ubiquitinated via transfer of a pre-formed ubiquitin chain on monoubiquitinated PCNA.


• Analyses of functional roles of TLS polymerases using transgenic mice

  HANAOKA Fumio, YOKOI Masayuki, AKAGI Jun-ichi

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A), Grant-in-Aid for Scientific Research (A), Gakushuin University, Apr. 2010 - Mar. 2013, Coinvestigator not use grants

  In order to examine the functional roles of translesion synthesis (TLS) in mammalian cells, we have established many mouse embryonic fibroblast cell lines expressing polymerase activity-defective or Rev1 interaction-defective mutant DNA polymerase eta (Pol eta) in Pol eta, Pol iota or Pol kappa single-, double- or triple-deficient mouse embryonic fibroblasts (MEFs). Using these MEF cells, we investigated UV sensitivity as well as mutation frequency upon UV irradiation. Our results suggest that there is an error-prone TLS pathway that uses Pol kappa via Rev1 in the absence of Pol eta.


• Physiological role of protein-protein interaction among translesion DNA polymerases in cellular resistance to ultraviolet light

  YOKOI Masayuki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Gakushuin University, Apr. 2010 - Mar. 2012, Principal investigator

  DNA polymerase η (Polη), whose gene mutation is responsible for the inherited disorder xeroderma pigmentosum variant (XP-V), carries out accurate and efficient TLS against cyclobutane pyrimidine dimer (CPD). Since Polη interacts with Polι and REV1, it is suggested that Polη plays 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 inactive Polη. Moreover, this recovery of cellular UV sensitivity was mediated by the interaction between Polη and REV1. However, expression of inactive mutant Polη was not able to suppress the striking incidence of UV-induced mutation observed in Polη-deficient cells. Finally we demonstrated that REV1 and Polκ are involved in mutagenic DNA damage tolerance via Polη-REV1 interaction when Polη was failed to bypass its cognate substrates.


• 色素性乾皮症バリアント群細胞とDT40細胞を用いた損傷乗り越え複製機構の解析

  横井 雅幸, 花岡文雄, 三木義男, 竹中克也

  東京医科歯科大学, 難治疾患共同研究拠点共同研究, 学習院大学, Apr. 2010 - Mar. 2011, Principal investigator


• Molecular mechanisms of translesion DNA synthesis and its involvement in carcinogenesis

  HANAOKA Fumio, YOKOI Masayuki, IWAI Shigenori

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas, Grant-in-Aid for Scientific Research on Priority Areas, Apr. 2005 - Mar. 2009, Coinvestigator

  We have clarified the mechanism in which human DNA polymerase η (Polη) interacts with other proteins for switching between Polη and the replicative polymerase at stalled replication forks. We have solved high-resolution crystal structures of human Polη at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. In addition, we have generated Polηknockout mice and observed high incidence of skin tumors upon UV-B irradiation in these mice.


• 転写因子の欠損に起因する発がんにおける基本転写因子の役割と核内シグナル伝達機構

  大熊 芳明, 横井 雅幸

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas, Grant-in-Aid for Scientific Research on Priority Areas, Osaka University, Apr. 2003 - Mar. 2004, Coinvestigator

  1.ヒトTFIIEαのZnフィンガーの機能解析 この領域の4個のシステインをアラニンに置換したC129A、C132A、C154A、C157Aと、グルタミン酸E140、アスパラギン酸D164をアラニン、リジンに置換したE140A、E140K、D164A、D164Kの点変異に関して、転写と他の基本転写因子との結合に関して解析した。その結果、ZnフィンガーのN末側は転写開始と伸長への移行の2段階に関わり、C末側は転写開始に機能する結果が得られた。他因子との相互作用を調べると、N末2変異はTFIIFβとの結合が強くなり、一方C末の2変異は、TFIIHのXPBサブユニットとの結合が減弱していたが、これらは転写結果を良く説明する。一方、酸性アミノ酸の点変異に関しては、転写がむしろ促進していた。特にD164Aは、転写活性が大きく増加してした。これら酸性領域には未知因子が機能している可能性が示唆され、今後これを検索していく。 2.分裂酵母TFIIEとRNAポリメラーゼII(PolII)の相互作用の解析 分裂酵母TFIIEの2サブユニットとPolIIサブユニットとの結合を調べたところ、TFIIEαはRpb5、βはRpb2とRpb12に強く結合した。PolIIは転写開始の際にその構造を変化させるが、その際に構造を変えるのは主にRpb5とRpb2である。これらに各々αとβサブユニットが結合することを我々が明らかにしたことで、転写の際にPolII活性中心近傍にTFIIEが結合するという事実と合わせ、この構造変化をTFIIE、特にそのαサブユニットが制御している可能性が考えられるに至った。 3.メディエーター複合体の解析 核内シグナル伝達に関わるメディエーターのサブユニットにタグを付け発現させるヒトHeLa細胞株を我々で確立し、これからメディエーターを精製したところ、3つのサブタイプからなることが分った。


• Studies on Molecular Mechanisms of Translesion Synthesis and Carcinogenesis

  HANAOKA Fumio, MASUTANI Chikahide, YOKOI Masayuki, OHKUMA Yoshiaki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas, Grant-in-Aid for Scientific Research on Priority Areas, Osaka University, Apr. 2000 - Mar. 2004, Coinvestigator not use grants

  In order to understand the molecular mechanisms of translesion synthesis and the relationships between carcinogenesis and translesion synthesis, we studied various aspects of translesion synthesis with special reference on human DNA polymerase η (Pol η) and obtained following results. 1. Human Pol η copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied. On the other hand, human Pol η catalyzes efficient and accurate translesion synthesis past cic-syn cyclobutane di-thymine lesions, AAF-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. 2. Human Pol η gene consists of 11 exons covering the entire coding region of the transcription-initiation, and the genomic DNA analysis suggested that three of four XP-V cell lines have homozygous mutations, and one of them have heterozygous point mutations, one of which is a splice mutation of the major allele. 3. Human Pol η binds template/primer DNAs regardless of TT dimers. Rather, enhanced binding to template/primer DNAs containing TT dimers is only observed when the 3-end of the primer is an adenosine residue situated opposite the lesion. When two nucleotides have been incorporated into the primer beyond the TT dimer position, the Pol η-template/primer DNA complex is destabilized. 4. In order to make a good in vivo model to study the high incidence of skin carcinogenesis in XP-V patients, we disrupted the mouse Pol η gene in ES cells by replacing exon 8 with the neo^r gene. Mice that are homozygous for the null mutation are viable, fertile and did not show any obvious defects during their first year of life. Pol η-deficient fibroblasts derived from the mutant mice have reduced capacity to replicate UV-damaged DNA. Pol η-deficient mice developed skin tumors after UVB-irradiation, while wild-type and heterozygous littermates did not.


• 複数のサイクリン依存性キナーゼ複合体によるRNAポリメラーゼIIの転写制御の解析

  大熊 芳明, 横井 雅幸

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas, Grant-in-Aid for Scientific Research on Priority Areas, Osaka University, Apr. 2002 - Mar. 2003, Coinvestigator

  1.RNAポリメラーゼII(PolII)の転写に機能するサイクリン依存性キナーゼの解析 PolIIのCTDの7アミノ酸リピート配列2番と5番セリンリン酸化は、PolIIに転写完結能を与えるのに重要である。最近は、RNAプロセシングやクロマチン修飾反応に関わる酵素もCTDのリン酸化セリン特異的に結合することで転写と協調的に制御されることが明らかになった。これらのPolIIリン酸化に関わるCDK/cyclin型キナーゼとして報告されたのは、TFIIH、メディエーター、転写伸長因子P-TEFbの3因子である。今年度は、メディエーターの転写における機能解析を進めると同時に、TFIIHサブユニットのTFIIEとの相互作用をヒトTFIIEαの点変異により解析した。TFIIEαZnフィンガーのXPBサブユニットとの結合結果は下に示すが、さらにC末の酸製領域がp62サブユニットと結合することを実証した。 2.ヒトTFIIEαのZnフィンガーの機能解析 この領域の4個のシステインをアラニンに置換したC129A、C132A、C154A、C157Aと、グルタミン酸E140、アスパラギン酸D164をアラニン、リジンに置換したE140A、E140K、D164A、D164Kの点変異に関して、転写と他の因子との結合に関して解析した。その結果、ZnフィンガーN末側は転写開始と伸長への移行の2段階に関わり、C末側は転写開始に機能する結果が得られた。他因子との相互作用を調べると、N末2変異はTFIIEβとの結合が強くなり、一方C末の2変異は、TFIIHのXPBサブユニットとの結合が減弱したが、これらは転写結果を良く説明する。一方、酸性アミノ酸の点変異に関しては、転写がむしろ促進していた。特にD164Aは、転写活性が大きく増加していた。これら酸性領域には未知因子が機能してしる可能性が示唆され、今後これを検索していく。


• 分裂酵母のDNA修復と損傷乗り越え複製制御機構の分子遺伝学的解析

  横井 雅幸

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A), Grant-in-Aid for Young Scientists (A), Osaka University, Apr. 2002 - Mar. 2003, Principal investigator

  (1)分裂酵母のDNA修復因子(ヌクレオチド除去修復、紫外線損傷除去修復関連因子)および損傷乗り越え複製活性を持つ複数のDNAポリメラーゼ(Eso1p、DinBp、Rev1p、Rev3p、Rev7p)に注目し、各遺伝子を破壊した株を樹立した。昨年度までに、各遺伝子破壊株の増殖能などは野生型並であることを報告している。 (2)作成した遺伝子破壊株の表現形解析を目的とし、紫外線および化学変異原に対する感受性を調査した。昨年度までに、一部の遺伝子破壊株における紫外線感受性実験の結果を報告している。まず、紫外線に対する多重遺伝子破壊株を用いた解析から、損傷乗り越え複製DNAポリメラーゼであるEso1pとRev(1,3,7)pの関わる経路が異なることを示す結果を得た。紫外線によるDNA損傷は主にシクロブタン型ピリミジン二量体(CPD)と(6-4)光産物であるが、それぞれを特異的に修復する酵素を分裂酵母細胞内で発現することで、Eso1pが主としてCPDの乗り越え反応に、またRev(1,3,7)pが主として(6-4)光産物の乗り越えに関わることが示された。一方、紫外線損傷と同様にして修復されるDNA損傷を引き起こす化学変異原である4NQOに対する感受性は、eso1遺伝子破壊株で最も顕著であり、また塩基損傷を引き起こすMMSに対する感受性はdinB遺伝子破壊株で高いことが示された。これらのことから、損傷の種類に応じた損傷乗り越えDNAポリメラーゼの使い分け機構の存在が遺伝学的解析によって示された。現在、上記の研究をさらに発展させると同時に、異なる化学変異原に対する感受性と、それに伴う突然変異発生率の測定を行っている。 (3)生化学的研究から、多様な損傷に対する損傷乗り越え複製の制御は、Rev1を中心としたTLSポリメラーゼ間の相互作用によるモデルが提唱されている。申請者は、一連のRev1P変異体をrev1遺伝子破壊株で発現させ、機能的相補実験を行った。その結果、Rev1pが正常に機能するためには、他のTLSポリメラーゼとの相互作用領域とTLS活性領域以外に、重要な役割を担う領域を見出した。この領域は、DNA修復やチェックポイント関連因子に見られるBRCTドメインを含み、今後、TLS機構とチェックポイントなどの細胞周期制御との関係を明らかにする上で貴重な情報である考えている。


• 転写因子の欠損に起因する発がんにおける基本転写因子の役割と核内シグナル伝達機構

  大熊 芳明, 横井 雅幸

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas, Grant-in-Aid for Scientific Research on Priority Areas, Osaka University, Apr. 2002 - Mar. 2003, Coinvestigator

  1.ヒトTFIIEαのZnフィンガー領域の構造解析 ヒト基本転写因子TFIIEはαとβサブユニットがα2β2の4量体を形成し、転写開始と伸長への移行に機能する。αは中央コア領域にZnフィンガーモチーフを有しており、この欠失変異体は試験管内転写系においてドミナントネガティブとして機能することから、転写に重要と考えられる。さらに真正細菌と真核生物の中間に位置する古細菌にもZnフィンガー領域を含むTFIIEαのN末端相当領域だけが保存されていることが示され、種を越えた機能の重要性が考えられている。そこで我々は、この領域をNMR構造解析し、亜鉛を配位するZnフィンガーモチーフであること、DNA結合型の従来のものと異なり表面は負電荷を帯びて蛋白相互作用に重要な役割を持つことを明らかにした。 2.RNAポリメラーゼII(PolII)の転写開始から伸長への移行段階の解析 TFIIEβサブユニットC末端ヘリックス領域に点変異を入れると、直鎖状DNA鋳型によるTFIIEの転写活性が失われた。これら変異は、TFIIHによるPolIICTDのリン酸化活性の促進能も失っていた。以上から、この領域は転写伸長への移行活性に関与すると考えられる。その機能メカニズムは、さらに検討中であるがTFIIHの転写伸長への移行に寄与するp44サブユニットとの結合が変異体では野生型より強くなっていたため、TFIIHとの作用の変化が寄与していると考えられる。 3.ヌクレオチド除去修復因子XPCの修復反応におけるTFIIHとの相互作用 紫外線等によるDNAの損傷の結果生じる皮膚癌を防ぐ損傷修復反応に関与する因子XPCは、遺伝病の色素性乾皮症原因因子で、損傷部位を認識して修復反応の引き金を引く因子であることを我々は明らかにしている。今回、XPCがそのC末端でTFIIHと結合することが修復活性においても大変重要であることを示した。


• STUDY ON NOVEL REQULATORY MECHANISMA OF DNA REPAIR

  HANAOKA Fumio, YOKOI Masayuki, MASUTANI Chikahide

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A), Grant-in-Aid for Scientific Research (A), OSAKA UNIVERSITY, Apr. 2001 - Mar. 2003, Coinvestigator not use grants

  In order to investigate how DNA repair systems are regulated, we analysed molecular mechanisms of nucleotide excision repair (NER) and translesion synthesis (TLS) in eukaryotic cells, and obtained the following results. 1) The XPC-HR23B protein complex, the initiator of global genome NER, was found to associate with a centrosome protein centrin 2. 2) The XPC-HR23B strongly recognized specific secondary structures of DNA, involving a single-and double-strand junction. A DNase I footprint analysis, using a looped DNA substrate, revealed that a single XPC-HR23B complex protected a distorted site in an asymmetrical manner. 3) While mHR23A KO mice showed no abnormalities, mHR23B KO mice showed impaired embryonic development and a high rate of intrauterine or neonatal death. Surviving animals display a variety of abnormalities, including retarded growth, facial dysmorphology, and male sterility. 4) XPC was found to interact with thymine DNA glycosylase (TDG) in yeast two hybrid screening. By biochemical analyses, XPC interacted with TDG not only physically but also functionally. 5) We identified two fission yeast homologs of budding yeast Rad4 and human XPC, designated Rfp4A and Rhp4B. Rhp4A was found to play roles in GGR and TCR, while Rhp4B acts as an accessory protein in GGR. 6) Human DNA polymerase η (Pol η) catalyzed relatively efficient and accurate TLS past 5R-thymineglycol and 5S-thymineglycol. 7) Both alleles of DNA polymerase τ (Pol τ) gene was found to have null mutation in 129-derived strains of mice. Overall frequency and spectrum of mutation were normal in Pol τ-deficient mice.


• DNA修復因子と中心体構成因子の遺伝的相互作用とその細胞生物学的意義

  横井 雅幸

  日本学術振興会, 科学研究費助成事業 萌芽研究, 萌芽研究, 大阪大学, 2002 - 2003

  (1)出芽酵母の中心体構成因子の一つCdc31に高い相同性を示す分裂酵母における相同因子Chp31(cdc31 homolog of S.pombe)の温度感受性変異株の作成を目的として、プラスミドシャッフリングを利用した変異株の単離・同定を試みているが温度感受性株を得るには至らなかった。chp31遺伝子の発現誘導をコントロールすることで、変異を導入したchp31遺伝子発現ライブラリーのスクリーニングを効率よく行うことを継続中である。その過程で、Chp31の過剰発現が増殖抑制をもたらす可能性を見出したが、その機構については現在調査中である。 (2)Rhp23は、分裂酵母のヌクレオチド除去修復(NER)因子である。Δrhp23株は、紫外線感受性を示すだけでなく、G2期の遅延や一部の細胞で染色体の不均等分配などが認められ、出芽酵母ホモログの解析からも中心体複製の過程に重要な役割を担うことが予想されている。一方でRhp23は、その分子内にユビキチン様構造(UbL)やユビキチン関連因子(UBA)との相同領域を持ち、ユビキチン蛋白質分解系への関与が知られている。我々のグループによるヒトホモログ(HR23B)の解析から、HR23Bにおける試験管内NER反応に必要な領域(NER活性ドメイン)は、これらとは異なる領域であることが明らかにされている。そこで、紫外線感受性を指標として、様々なRhp23変異体蛋白質を用いたNER活性ドメインの同定を行った。その結果、Rhp23のヒトホモログNER活性ドメイン相同部位を含む領域が、分裂酵母細胞内のNER活性に必要であることが示された。Rhp23は、Rhp41あるいはRhp42と複合体を形成してNERに参加すると考えられていることから、Rhp23における結合ドメインの探索を、大腸菌発現系を用いた組換え蛋白質を材料として行った。その結果、Rhp41とRhp42への結合は、Rhp23の細胞内におけるNER活性領域と重なることを明らかにした。また、rhp23-rhp41二重破壊株あるいは、rhp23-rhp42二重破壊株の紫外線感受性回復実験から、Rhp41と結合しNER活性を示すにはNER活性ドメインを含む領域だけで十分だが、Rhp42と結合し、かつNER反応で十分機能するには、少なくともRhp23のUBA領域が関わる可能性を示した。このことは、Rhp41とRhp42の機能に対するRhp23の果たす役割りが異なる可能性や、NER反応とユビキチン系蛋白質分解のクロストークの存在を示唆するもので、今後更なる解析を必要とする。加えて、UbLやUBA領域を持たない変異体ではΔrhp23株の示す細胞周期や染色体分配の異常が相補されないことから、これらの形質と蛋白質分解系の関連を解明することも重要である。


• 転写因子の欠損に起因する発がんにおける基本転写因子の役割と核内シグナル伝達機構

  大熊 芳明, 横井 雅幸

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas (C), Grant-in-Aid for Scientific Research on Priority Areas (C), Osaka University, Apr. 2001 - Mar. 2002

  1.ヒトTFIIEαのZnフィンガー領域の構造解析 ヒトTFIIEは、αとβの2サブユニットがα2β2の4量体を形成し、転写開始と伸長への移行に機能する。αは中央のコア領域にZnフィンガーモチーフを有しており、この欠失変異体は試験管内転写系においてドミナントネガティブとして機能することから、転写において重要な機能を担っていると考えられる。さらに原核生物である古細菌類にも最近、ヒトTFIIEαのZnフィンガー領域を含むN末端領域だけがホモログとして存在することが示され、その種を越えた機能の重要性が考えれている。そこで我々は、この領域をNMRにより構造解析した。その結果、亜鉛依存に構造を形づくる、Znフィンガーモチーフであることが実証された。その機能について、現在解析中である。 2.RNAポリメラーゼII (Pol II)の転写開始から伸長への移行段階の解析 昨年度、線虫TFIIEβホモログを用いて、Pol II最大サブユニットC末端の7アミノ酸繰り返しCTD配列の2番目と5番目のセリンのTFIIHによるリン酸化のうち、5番目のセリンリン酸化が転写伸長への移行活性と密接な関連性があることを示してきた。そこで、今年度はさらにβサブユニットのC末端にTFIIEのこの転写伸長への移行活性に関与する領域があることを見いだした。この領域内のアミノ酸残基を点突然変異により置換すると移行活性のみならず、Pol IIリン酸化活性も大きく低下した。 3.ヌクレオチド除去修復因子XPCの修復反応におけるTFIIHとの相互作用 紫外線等によるDNAの損傷の結果生じる皮膚癌を防ぐ損傷修復反応に関与する因子XPCは、損傷部位を認識して修復反応の引き金を引く因子であることを我々は明らかにしている。今回このXPCがそのC末端でTFIIHと結合することが修復活性においても大変重要であることを示した。


• Biochemical and Ganetical Analyses of Organized Regularly Mechanism of Gene Expression by Various Transcription Super-Complex.

  OHKUMA Yoshiaki, EKI Toshihiko, YOKOI Masayuki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Osaka university, 2001 - 2002, Coinvestigator not use grants

  1. Characterization of the Mediator complex. The Mediator consists of more than 20 subunits and transmits the transcriptional regulation signals from outsaide of the cell nucleus to RNA rolymersae 11 (Pol II). We tagged three subunits independently, established HeLa cell lines expressing each tagged-subunit, and purified the complexes from nuclear extracts. As a result, one transcription super-complex with molecular mass 1-2 MDa was purified from all cell lines and showed positive effects on transriptionnal activation. 2. Analyses of the Zn finger of TFIIEα. Human general transcription factor TFIIE consists of α and β subunits, froms an α2 β2 tetramer, and functions at transcription initiation and the transition to elongation. TFIIE α possesses a Zn finger motif at the central core region and this might be important for transcription system. Moreover, it was demonstrated that the N-terminal region of TFIIEα including this motif is conserved in Archaebacteria classifies between prokaryotes and eukaryotes. We studied this region by NMR and found that this actually forms the Zn finger motif, and that region has important roles in protein-protein interactions by negative charges on its surface. 3. Studies on the transition of Pol II from initiation to elongation. By point mutations in the C-terminal helix region of TFIIEβ, the transcriptional activity with a linearized template has been lost. These mutants lost the stimulation activity of TFIIH-mediated phosphorylation of Pol II. From these results, this region might be involved in the transition. functional mechanisms are still elucive, but it is highly conceivable that the changes in interactions between TFIIE and TFIIH are involved, since the binding of p44 of TFIIH, which is essential for the transition to elongation, to those mutants became stronger than that to the wild type.


• 真核生物のヌクレオチド除去修復の解析とモデル動物を用いた発癌機構の研究

  横井 雅幸

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Young Scientists (A), Grant-in-Aid for Encouragement of Young Scientists (A), Osaka University, Apr. 2000 - Mar. 2001, Principal investigator

  1.分裂酵母のヌクレオチド除去修復(NER)機構の解析 ヒトXPC/出芽酵母Rad4のホモログが分裂酵母に2種類(Rhp4A、Rhp4B)存在することを証明し、それらのNER機構での役割を解析した。生化学的解析から、Rhp4A及び4Bの両者はヒトXPC/出芽酵母Rad4と同様に、損傷DNAに対して高い親和性を示すことを明らかにした。また、遺伝学的解析から、Rhp4Aは出芽酵母Rap4と同様、ゲノム全体の修復(GGR)と転写と共役した修復(TCR)の両方で機能するのに対し、Rhp4BはヒトXPCと同じく、GGRにだけ機能することを明らかにした(論文投稿中)。 この研究は、なぜヒトXPCがGGR特異的であるか、またTCRでのDNA損傷認識過程を解析する上で重要である。さらに、Rhp4A及び4Bと複合体を形成し、分裂酵母のNER因子として働くRhp23の遺伝学的解析から、Rhp23が細胞周期制御に関わることを示唆する結果を得た。現在、DNA修復と細胞周期チェツクポイントに注目し、研究している。 2.XPCノックアウトマウスの作成と解析 XPCの機能を個体レベルで解析する目的で、モデル動物としてノックアウトマウスを作出した。その際、XPC遺伝子をβガラクトシダーゼ(β-gal)遺伝子で置き換え、XPC遺伝子の発現をβ-galの活性を指標として検出できるように工夫した。.マウス胎児から樹立した胚性細胞を用いた解析から、XPC遺伝子を欠失した細胞では紫外線高感受性になること、またβ-galの活性の変動によりXPC遺伝子の発現が紫外線照射により有意に上昇することを明らかにした。さらに、発生段階の異なるXPC(+/-)マウス胚でβ-galの発現パターンを調べた結果、脳の特定の部位でXPC遺伝子の高い発現が認められた。現在、個体レベルでの発癌機構の解析と平行し、XPC遺伝子の発現が高い部位の特定とその生理学的意義を明らかにしょうとしている。