Elucidation of mechanisms that the eukaryotic mismatch repair system regulates PCNA unloading

• Project/Area Number: 17H06935
• Research Category: Grant-in-Aid for Research Activity Start-up
• Allocation Type: Single-year Grants
• Research Field: Molecular biology
• Research Institution: Kyushu University
• Principal Investigator: Kawasoe Yoshitaka 九州大学, 理学研究院, 学術研究員 (60805422)
• Project Period (FY): 2017-08-25 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: PCNA / ミスマッチ修復 / DNA複製 / ツメガエル卵抽出液 / クランプローダー / PCNAアンローディング

Outline of Final Research Achievements

The replication clamp PCNA is essential for various reactions coupled with DNA replication. PCNA is loaded onto DNA by clamp loader complexes, and DNA-loaded PCNA functions as a platform for numerous factors involved in DNA replication and repair. We previously found that the unloading of PCNA is regulated by the mismatch recognition complex MutSα. In this study, we examined the dynamics of PCNA unloading in Xenopus egg extracts and a reconstitution system with purified proteins. Our results indicate that the Elg1-RFC complex unloads PCNA in Xenopus egg extracts and suggest a possibility that an additional factor(s) is involved in the Elg1-RFC-mediated PCNA unloading reaction.

Academic Significance and Societal Importance of the Research Achievements

PCNAのDNA上でのローディングやアンローディングは、PCNAを足場する様々な反応を協調的に機能させ、ゲノムを安定に維持するために非常に厳密に制御される必要がある。実際に酵母ではElg1の欠失によって突然変異や染色体再編の頻度が上昇し、哺乳類においても多くの臓器での腫瘍形成が報告されている。PCNAアンローディング反応のみに着目した申請者独自の生化学的解析によって得られた本成果は、生化学的な知見が乏しいアンローディング過程の理解を深め、PCNAのDNA上での挙動制御の理解が進むだけではなく、人工的な変異導入法、がんや遺伝病などの原因解明、創薬のターゲットの応用へと繋がると期待される。

Research Products

• [Journal Article] Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1. (2018)
  • Author(s): Terui R, Nagao K, Kawasoe Y, Taki K, Higashi TL, Tanaka S, Nakagawa T, Obuse C, Masukata H, Takahashi TS
  • Journal Title: Genes & development Volume: 32(11-12) Issue: 11-12 Pages: 806-821
  • DOI: 10.1101/gad.310995.117
  • Peer Reviewed / Open Access
• [Journal Article] Sensing and Processing of DNA Interstrand Crosslinks by the Mismatch Repair Pathway. (2017)
  • Author(s): Kato N, Kawasoe Y, Williams H, Coates E, Roy U, Shi Y, Beese LS, Scharer OD, Yan H, Gottesman ME, Takahashi TS, Gautier J.
  • Journal Title: Cell Reports Volume: 21 Issue: 5 Pages: 1375-1385
  • DOI: 10.1016/j.celrep.2017.10.032
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Elg1-Rfc2-5複合体（Elg1-RFC）はツメガエル卵抽出液における主たるPCNAアンローディング複合体である (2018)
  • Author(s): 河添好孝、下川紗貴子、釣本敏樹、高橋達郎
  • Organizer: 第41回日本分子生物学会年会
• [Presentation] The Elg1-Rfc2-5 complex (Elg1-RFC) is the major PCNA-unloading complex in Xenopus egg extracts (2018)
  • Author(s): Yoshitaka Kawasoe、Sakiko Shimokawa、Toshiki Tsurimoto、Tatsuro Takahashi
  • Organizer: OKAZAKI Fragment Memorial Symposium: Celebrating the 50th anniversary of the discontinuous DNA replication model
  • Int'l Joint Research
• [Presentation] ミスマッチ修復経路に依存した抗組換え反応の試験管内再現による解析 (2017)
  • Author(s): 織田 里美、河添 好孝、坂詰 彩、中川 拓郎、升方 久夫、高橋 達郎
  • Organizer: 2017年度生命科学系学会合同年次大会