Time series visualization of DNA replication initiation process by protein X-ray crystallography

• Project/Area Number: 25840027
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: National Institute of Genetics
• Principal Investigator: ITOU Hiroshi 国立遺伝学研究所, 細胞遺伝研究系, 特任研究員 (10390626)
• Research Collaborator: ITOH Tateo 信州大学, 理学部, 特任教授 (40051817)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2013: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: DNA複製開始因子 / タンパク質DNA複合体 / プラスミド / Rep / PriCT / X線結晶構造解析法 / PriCT領域 / X線結晶構造解析 / タンパク-DNA複合体

Outline of Final Research Achievements

Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2 is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. We determined the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three structural modules. The structure also shows that the functionally unknown PriCT domain plays a central role in DNA unwinding. The conservation of the PriCT domain in the C-termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. This is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.

Research Products

• [Journal Article] Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: Duplex DNA unwinding by a single protein (2015)
  • Author(s): H.Itou, M.Yagura, Y.Shirakihara, T.Itoh
  • Journal Title: Journal of Biological Chemistry Volume: 290 Issue: 6 Pages: 3601-6311
  • DOI: 10.1074/jbc.m114.595645
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Structural basis of replication origin binding and double-stranded DNA unwinding by an initiator-primase ColE2-Rep protein (2013)
  • Author(s): Hiroshi Itou, Masaru Yagura, Tateo Itoh and Yasuo Shirakihara
  • Organizer: International Conference on Structural Genomics 2013
  • Place of Presentation: 北海道、札幌市、京王プラザホテル札幌