Electron microscopic analysis of a G4 DNA-binding protein Rif1, a key organizer of chromosomal domains

2020 Fiscal Year Final Research Report

• Project/Area Number: 18K06102
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: MORIYAMA Kenji 公益財団法人東京都医学総合研究所, 基礎医科学研究分野, 研究員 (00250217)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Keywords: Rif1 / タンパク質精製 / オリゴマー / グアニン四重鎖 / G4 DNA / 電子顕微鏡 / 単粒子解析

Outline of Final Research Achievements

I made enormous efforts to purify 2,418 amino acid (a.a.)-long murine Rif1 to analyze molecular shapes of its oligomer(s) and its complex with G-quadruplex (G4) DNA via single particle analyses. However, amount of the purified Rif1 was not enough to electron microscopic (EM) analysis. Thus, I next purified partially-truncated Rif1 protein, termed Rif1-NC, lacking a long IDP (intrinsically disordered polypeptide) segment (968 a.a.) present between its NTD and CTD. EM analyses of the purified Rif1-NC verified its oligomeric nature, but failed to unveil its molecular shape in high resolution. Then, I turned my plan toward EM analysis of fission yeast Rif1 from Schizosaccharomyces pombe (SpRif1), because of its natively short length (1,400 a.a.). It was difficult to purify full-length SpRif1, but I succeeded to purify it after short deletion of an N-terminal 92 a.a. (thus, termed as SpRif1dN92). The EM analysis is now in progress with the purified SpRif1dN92 protein.

Free Research Field

生物化学

Academic Significance and Societal Importance of the Research Achievements

研究代表者の先行研究の中で、Rif1が複数のG4（グアニン四重鎖）DNA結合部位を持つオリゴマーであることを生化学的に示してはいたが、今回の電子顕微鏡解析によりRif1がオリゴマーであることを視覚的に裏付けることができたのは学術的に意義深い。また、本研究は米国のクライオ電顕解析のエキスパートであるHuilin Li教授達（Van Andel Research Institute）との国際共同研究であり、科学的な国際強調の観点でその社会的意義は大きい。