Comprehensive analysis of mechanisms for stalled replication fork stabilization by quantitative proteomics

• Project/Area Number: 16K07252
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Molecular biology
• Research Institution: Kyoto University
• Principal Investigator: Yamada Masayuki 京都大学, 医学研究科, 講師 (40398798)
• Project Period (FY): 2016-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: Cdc7 / ユビキチン化 / 脱ユビキチン化 / replisome / 複製ストレス / Cdc7 kinase / replisome stability / deubiquitinase / replication stress / replication fork / Cdc7-ASK/Dbf4 kinase / protein stability / proteomics / 複製フォーク / Chk1 / プロテオミクス / ゲノム安定性 / Cdc7キナーゼ

Outline of Final Research Achievements

Cdc7 protein functions as a protein kinase which plays an important role in DNA replication. Small-molecule inhibitors of Cdc7 kinase are currently expected as novel anticancer drugs. Cdc7 kinase activity is positively regulated by binding to ASK protein. The molecular mechanism by which ASK protein is stabilized/destabilized is still unknown. In this research project, I found that a deubiquitinase USP7 stabilizes ASK protein, which leads to activation of Cdc7 kinase. In addition, I also found that Cdc7 kinase inhibition induces replication fork collapses caused by dissociation of some proteins of DNA replication machinery.

Academic Significance and Societal Importance of the Research Achievements

数多くの国内外の研究グループによる最近の研究により、がん遺伝子の活性化やがん抑制遺伝子の不活性化による無秩序な増殖シグナルがもたらす制御を逸脱したDNA複製が複製ストレスを引き起こし、それがゲノムの不安定性につながると考えられるようになってきた。本研究は、複製ストレス下では複製フォーク近傍でどのようなことが起こっているかの一端を明らかにした。
さらに、多くの抗がん剤はDNA複製を阻害する作用があることから、抗がん剤によるDNA複製阻害時に複製フォーク上で何が起こっているのかを明らかにする端緒となり、現在開発が進んでいるCdc7阻害剤を用いた新たな化学療法の開発に向けた貴重な基礎データとなる。

Research Products

• [Int'l Joint Research] Danish Cancer Society(デンマーク)
• [Int'l Joint Research] Palacky University(チェコ)
• [Int'l Joint Research] Karolinska Institute(スウェーデン)
• [Int'l Joint Research] Danish Cancer Society Research Center(Denmark)
• [Int'l Joint Research] Karolinska Institute(Sweden)
• [Int'l Joint Research] Palacky University(チェコ)
• [Int'l Joint Research] Danish Cancer Society Research Center(Denmark)
• [Int'l Joint Research] Palacky University(チェコ)
• [Int'l Joint Research] Karolinska Institute(Sweden)
• [Journal Article] Cdc7 kinase stimulates Aurora B kinase in M-phase (2019)
  • Author(s): Ito Sayuri、Goto Hidemasa、Kuniyasu Kinue、Shindo Mayumi、Yamada Masayuki、Tanaka Kozo、Toh Gaik-Theng、Sawa Masaaki、Inagaki Masaki、Bartek Jiri、Masai Hisao
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 18622-18622
  • DOI: 10.1038/s41598-019-54738-2
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL (2018)
  • Author(s): Morichika Takita, Fujiko Tsukahara, Taishi Mishima, Katsuaki Ieguchi, Masayuki Yamada, Hiroaki Honda, and Yoshiro Maru
  • Journal Title: Oncotarget Volume: 9(60) Issue: 60 Pages: 31682-31696
  • DOI: 10.18632/oncotarget.25849
  • Peer Reviewed / Open Access
• [Journal Article] C1D is not directly involved in the repair of UV-damaged DNA but protects cells from oxidative stress by regulating gene expressions in human cell lines. (2018)
  • Author(s): Tomita T, Ieguchi K, Takita M, Tsukahara F, Yamada M, Egly JM, Maru Y.
  • Journal Title: J Biochem Volume: 164 Pages: 415-426
  • DOI: 10.1093/jb/mvy069
  • NAID: 40021789520
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Role of DNA repair factor XPC in response to replication stress, revealed by DNA fragile site affinity chromatography and quantitative proteomics (2016)
  • Author(s): Beresova L, Vesela E, Chamrad I, Voller J, Yamada M, Furst T, Lenobel R, Chroma K, Gursky J, Krizova K, Mistrik M, Bartek J.
  • Journal Title: Journal of Proteome Research Volume: 15 Issue: 12 Pages: 4505-4517
  • DOI: 10.1021/acs.jproteome.6b00622
  • Peer Reviewed / Int'l Joint Research