Functional analyses and development of inhibitors based on higher-order structure of translesion DNA polymerase eta

• Project/Area Number: 15H04646
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: Gakushuin University
• Principal Investigator: HANAOKA Fumio 学習院大学, 理学部, 研究員 (50012670)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥16,900,000 (Direct Cost: ¥13,000,000、Indirect Cost: ¥3,900,000); Fiscal Year 2017: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2016: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2015: ¥8,710,000 (Direct Cost: ¥6,700,000、Indirect Cost: ¥2,010,000)
• Keywords: DNAポリメラーゼ / 損傷乗り越え合成 / DNA損傷 / 紫外線感受性 / 突然変異 / 阻害剤 / スクリーニング / 遺伝子 / 癌 / CPD

Outline of Final Research Achievements

In order to identify the important amino acid residues for the template DNA binding of human DNA polymerase eta (Pol eta), we generated several point mutants of Pol eta based on the co-crystal structure of Pol eta-UV-damaged DNA complex. We analyzed these purified Pol eta biochemically as well as UV sensitivity and mutation frequency of 6-thioguanine resistance.
We also conducted screening of Pol eta inhibitors using chemical arrays of natural low molecular-weight compounds by Pol eta-binding at first and then examined inhibitory activities of these compounds using primer-extension reaction. We successfully obtained several Pol eta inhibitor candidates and some of them are plausibly specific for Pol eta.

Academic Significance and Societal Importance of the Research Achievements

ヒトPolηと紫外線損傷DNAとの共結晶構造に基づいて、鋳型DNAとの相互作用に重要と考えられるアミノ酸を置換した点変異体タンパク質の解析から、鋳型DNAとの相互作用が弱い変異体は全て野生型と比較してDNA鎖の伸長反応が進みにくい結果となった。このことは結晶構造解析によるタンパク質の機能予測が非常に的確であることを示している。
一方、天然物由来の低分子化合物を固定化した化合物アレイを用い、ヒトPolηに対して阻害効果を示す化合物のスクリーニングを行い、Polηに特異的な阻害剤の候補を選択した。これらは代表的な制がん剤であるシスプラチンとの併用により、制がん効果の増強に効果的な可能性がある。

Research Products

• [Journal Article] Polη, a Y-family translesion synthesis polymerase, promotes cellular tolerance of Myc-induced replication stress (2018)
  • Author(s): Kurashima Kiminori、Sekimoto Takayuki、Oda Tsukasa、Kawabata Tsuyoshi、Hanaoka Fumio、Yamashita Takayuki
  • Journal Title: Journal of Cell Science Volume: 131 Issue: 12
  • DOI: 10.1242/jcs.212183
  • Peer Reviewed / Open Access
• [Journal Article] Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites (2018)
  • Author(s): Yasuda Takeshi、Kagawa Wataru、Tajima Katsushi
  • Journal Title: PLOS Genetics Volume: 14 Issue: 3 Pages: 1007277-1007277
  • DOI: 10.1371/journal.pgen.1007277
  • NAID: 120006459820
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 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 (2018)
  • Author(s): Akagi Jun-ichi、Yokoi Masayuki、Cho Young-Man、Toyoda Takeshi、Ohmori Haruo、Hanaoka Fumio、Ogawa Kumiko
  • Journal Title: DNA Repair Volume: 61 Pages: 76-85
  • DOI: 10.1016/j.dnarep.2017.11.006
  • NAID: 120006456929
  • Peer Reviewed / Open Access
• [Journal Article] Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA superhelical state. (2017)
  • Author(s): Takahashi S, Motooka S, Kawasaki S, Kurita H, Mizuno T, Matsuura SI, Hanaoka F, Mizuno A, Oshige M, Katsura S
  • Journal Title: J Biomol Struct Dyn Volume: 5 Issue: 1 Pages: 1-13
  • DOI: 10.1080/07391102.2016.1269689
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing (2017)
  • Author(s): Izumi Masako、Mizuno Takeshi、Yanagi Ken-ichiro、Sugimura Kazuto、Okumura Katsuzumi、Imamoto Naoko、Abe Tomoko、Hanaoka Fumio
  • Journal Title: Journal of Biological Chemistry Volume: 292 Issue: 31 Pages: 13008-13021
  • DOI: 10.1074/jbc.m117.779371
  • Peer Reviewed / Open Access
• [Journal Article] Two mammalian homologs of yeast Rad23, HR23A and HR23B, as multifunctional proteins. (2017)
  • Author(s): Yokoi M, Hanaoka F
  • Journal Title: Gene Volume: 597 Pages: 1-9
  • DOI: 10.1016/j.gene.2016.10.027
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] 2,2,4-Triamino-5(2H)-oxazolone is a weak substrate for nucleotide excision repair. (2016)
  • Author(s): Kino K, Sugasawa K, Miyazawa H, Hanaoka F
  • Journal Title: J Pharm Negative Results Volume: 7 Issue: 1 Pages: 42-45
  • DOI: 10.4103/0976-9234.177068
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι (2015)
  • Author(s): Kanao R, Yokoi M, Ohkumo T, Sakurai Y, Dotsu K, Kura S, Nakatsu Y, Tsuzuki T, Masutani C, Hanaoka F
  • Journal Title: DNA Repair (Amsterdam) Volume: S1568-7864(15) Pages: 00042-7
  • DOI: 10.1016/j.dnarep.2015.02.006
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions (2015)
  • Author(s): Anastasia Tsaalbi-Shtylik, Cristina Ferrás, Bea Pauw, Giel Hendriks, Piya Temviriyanukul, Leone Carlée, Fabienne Calléja, Sandrine van Hees, Jun-Ichi Akagi, Shigenori Iwai, Fumio Hanaoka, Jacob G. Jansen, and Niels de Wind
  • Journal Title: The Journal of Cell Biology Volume: 209 Issue: 1 Pages: 33-46
  • DOI: 10.1083/jcb.201408017
  • Peer Reviewed / Open Access
• [Journal Article] SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair (2015)
  • Author(s): Akita M
  • Journal Title: Sci. Rep. Volume: 5 Issue: 1 Pages: 10984-10984
  • DOI: 10.1038/srep10984
  • Peer Reviewed / Open Access
• [Journal Article] The protein level of Rev1, a TLS polymerase in fission yeast, is strictly regulated during the cell cycle and after DNA damage. (2015)
  • Author(s): Uchiyama M, Terunuma J, Hanaoka F
  • Journal Title: PLoS One Volume: 10 Issue: 7 Pages: e0130000-e0130000
  • DOI: 10.1371/journal.pone.0130000
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases. (2015)
  • Author(s): Masuda Y, Kanao R, Kaji K, Ohmori H, Hanaoka F, Masutani C
  • Journal Title: Nucleic Acids Res Volume: 43 Issue: 16 Pages: 7898-7910
  • DOI: 10.1093/nar/gkv712
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural basis of pyrimidine-pyrimidone (6-4) photoproduct recognition by UV-DDB in the nucleosome. (2015)
  • Author(s): Osakabe, A., Tachiwana, H., Kagawa, W., Horikoshi, N., Matsumoto, S., Hasegawa, M., Matsumoto, N., Toga, T., Yamamoto, J., Hanaoka, F., Thoma, N.H., Sugasawa, K., Iwai, S., Kurumizaka, H.
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 16330-16330
  • DOI: 10.1038/srep16330
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] USP7 is a suppressor of PCNA ubiquitination and oxidative-stress-induced mutagenesis in human cells. (2015)
  • Author(s): Kashiwaba S, Kanao R, Masuda Y, Kusumoto-Matsuo R, Hanaoka F, Masutani C
  • Journal Title: Cell Rep Volume: 13 Pages: 2072-2080
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] C57BL/6J野生型およびPolκ欠損マウスにおけるベンゾ[a]ピレンおよびα‐ナフトフラボン併用投与の効果 (2019)
  • Author(s): 赤木純一, CHO Young‐Man, 豊田武士, 横井雅幸, 花岡文雄, 大森治夫, 小川久美子
  • Organizer: 第35回日本毒性病理学会学術総会
• [Presentation] N7グリシドアミドdG付加体は哺乳類細胞においてDNA複製を阻害する (2018)
  • Author(s): 赤木純一, 横井雅幸, Young-Man Cho, 岩井成憲, 花岡文雄, 小川久美子
  • Organizer: 第41回日本分子生物学会年会（2018年11月28日、横浜）
• [Presentation] Benzo[a]pyrene-induced tumorigenesis in Polκ-knockout mice (2018)
  • Author(s): Jun-ichi Akagi, Young-Man Cho, Takeshi Toyoda, Masayuki Yokoi, Fumio Hanaoka, Haruo Ohmori, Kumiko Ogawa
  • Organizer: The 11th 3R & 3C Symposium
  • Int'l Joint Research
• [Presentation] Effect of Polκ deficiency on benzo[a]pyrene-induced tumorigenesis (2018)
  • Author(s): Jun-ichi Akagi, Young-Man Cho, Takeshi Toyoda, Masayuki Yokoi, Fumio Hanaoka, Haruo Ohmori, Kumiko Ogawa
  • Organizer: 5th DNA Polymerases meeting
  • Int'l Joint Research
• [Presentation] ベンゾ[a]ピレン誘発発がんに対するPolκの寄与の解析 (2018)
  • Author(s): 赤木純一, Young-Man Cho, 豊田武士, 水田保子, 横井雅幸, 大森治夫, 花岡文雄, 小川久美子
  • Organizer: 日本薬学会第138年会
• [Presentation] ベンゾ[a]ピレン混餌投与によるマウス前胃腫瘍発生に対するPolκの寄与 (2017)
  • Author(s): Jun-ichi Akagi, Young-Man Cho, Takeshi Toyoda, Yasuko Mizuta, Masayuki Yokoi, Fumio Hanaoka, Haruo Ohmori, Kumiko Ogawa
  • Organizer: 2017年度生命科学系学会合同年次大会
• [Presentation] ヒトMCM10のMCM2-7相互作用ドメインの同定 (2017)
  • Author(s): 泉雅子、水野武、柳憲一郎、杉村和人、奥村克純、今本尚子、阿部知子、花岡文雄
  • Organizer: 第24回DNA複製・組換え・修復ワークショップ
• [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酵素とクロマチン構造変換因子の探索 (2017)
  • Author(s): 横井雅幸、鹿谷有由希、菅澤薫、花岡文雄
  • Organizer: 第24回DNA複製・組換え・修復ワークショップ
• [Presentation] TLS polymerases and mutagenesis (2017)
  • Author(s): Fumio Hanaoka
  • Organizer: ICEM-ACEM 2017 Satellite Symposium on DNA Repair
  • Int'l Joint Research / Invited
• [Presentation] シスプラチン処理した細胞においてヒトDNAポリメラーゼηの働きに寄与するクロマチン構造変換因子 (2017)
  • Author(s): 横井雅幸、菅澤薫、花岡文雄
  • Organizer: 第76回日本癌学会学術総会
• [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 (2017)
  • Author(s): 赤木純一, 横井雅幸, Young-Man Cho, 豊田武士, 大森治夫, 花岡文雄, 小川久美子
  • Organizer: 第３回次世代を担う若手のためのレギュラトリーサイエンスフォーラム
• [Presentation] My CV: From our studies on XP-C to XP-V. 6th US-Japan DNA Repair Meeting (2017)
  • Author(s): Fumio Hanaoka
  • Organizer: 6th US-Japan DNA Repair Meeting
  • Int'l Joint Research / Invited
• [Presentation] Mutant analyses of human DNA polymerase eta based on high-resolution crystal structures of the protein-DNA-dNTP complex. (2016)
  • Author(s): Hanaoka F, Yokoi M, Masutani C, Yang W
  • Organizer: 4th International Meeting on DNA Polymerases.
  • Place of Presentation: Biarritz, France
  • Year and Date: 2016-10-04
  • Int'l Joint Research / Invited
• [Presentation] Cancer and translesion synthesis polymerases. (2016)
  • Author(s): Sakurai Y, Yokoi M, Hanaoka F
  • Organizer: 2016 Cold Spring Harbor Asia Conference-DNA Metabolism, Genomic Stability and Diseases
  • Place of Presentation: Suzhou, China
  • Year and Date: 2016-06-13
  • Int'l Joint Research / Invited
• [Presentation] Pol eta, Pol iota, and Pol kappa exhibit different genetic interactions to various compounds of different genotoxic mechanisms and the triple knockout cells are useful for screening of genotoxins. (2016)
  • Author(s): Akagi J, Yokoi M, Toyoda T, Cho YM, Ohmori H, Hanaoka F, Ogawa K
  • Organizer: Gordon Research Conference on Mutagenesis
  • Place of Presentation: Girona, Spain
  • Year and Date: 2016-06-05
  • Int'l Joint Research
• [Presentation] 私のDNA修復研究： XPCからXPVへ (2015)
  • Author(s): 花岡文雄
  • Organizer: 新学術領域研究「ゲノム普遍的制御」（H22～H26年度）公開シンポジウム『ゲノム安定性の機構と生命の維持ー進化、癌化、老化の理解のために』
  • Place of Presentation: 京都大学
  • Year and Date: 2015-08-28
  • Invited
• [Presentation] Regulation of the protein level of Rev1 in fission yeast. (2015)
  • Author(s): Uchiyama M, TerunumaJ, Hanaoka F
  • Organizer: Zing Genomic Integrity Conference 2015
  • Place of Presentation: Cairns, Australia
  • Year and Date: 2015-08-01
  • Int'l Joint Research / Invited
• [Presentation] Forty years of DNA damage tolerance. (2015)
  • Author(s): Hanaoka F
  • Organizer: 15th International Congress of Radiation Research (ICRR 2015)
  • Place of Presentation: 京都国際会館
  • Year and Date: 2015-05-25
  • Int'l Joint Research / Invited
• [Book] DNA Replication, Recombination, and Repair (2016)
  • Author(s): Hanaoka F, Sugasawa K
  • Total Pages: 555
  • Publisher: Springer Japan
• [Book] Function of translesion DNA polymerases in genome stability (2015)
  • Author(s): Masutani C, Kanao R, Hanaoka F
  • Total Pages: 260
  • Publisher: Research Signpost