Molecular Mechanisms of recognition of G-quadruplex by Rif1, a conserved nuclear factor regulating chromatin architecture

2021 Fiscal Year Final Research Report

• Project/Area Number: 19K16082
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 43030:Functional biochemistry-related
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: KAKUSHO Naoko 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 (30599593)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: グアニン４重鎖DNA / Rif1タンパク質 / クロマチンループ / 高次構造 / 複製タイミング / RNA-DNAハイブリッド / 分裂酵母 / 多量体形成

Outline of Final Research Achievements

The evolutionally conserved Rif1 protein binds to chromatin through recognizing G4 structure in fission yeast, and regulates replication timing by suppressing origin activation. The C-terminal segment of Rif1 contains G4 binding and oligomerization activities and we have identified critical residues for each function. We then showed both functions are independently required for Rif1-mediated replication inhibition. We have shown that the C-terminal 229 aa exhibits G4 binding activity in gel shift assays, while the C-terminal 44 aa polypeptide shows oligomerization activity. The C-terminal segment was shown to be a tetramer by SEC-MALS analyses. Structure prediction strongly suggests that the C-terminal segment adopts an amphipathic coiled-coil structure. The mutations were predicted to disrupt the stability of coiled-coil and their oligomerization.

Free Research Field

分子生物学

Academic Significance and Societal Importance of the Research Achievements

Rif1は、染色体の高次構造の形成を介して、複製や修復などクロマチン機能を制御すると考えられる。また、Rif1はG4構造に特異的に結合する。G4はヒト細胞内の染色体上に、実際に120,000個以上存在し、種々のゲノム機能に重要な役割を果たすことが示唆されており、G4の核内動態とその結合タンパク質による認識機構は、最近大きな注目を集めている。Rif1-G4相互作用とそれによる複製制御をモデルとした本研究は、ゲノムの未知のシグナルの解明、及びその疾患との関連に新たな洞察を与える。