1 Structure and mutagenesis studies of the C-terminal region of licensing factor Cdt1 enable to identify the key residues for binding to replicative helicase Mcm proteins. JunGoo Jee ¶, * ,1 , Takeshi Mizuno §, * ,√ , Katsuhiko Kamada §,+ , Hidehito Tochio † , Yasumasa Chiba & , Ken-ichiro Yanagi § , Gentaro Yasuda § , Hidekazu Hiroaki ‡ , Fumio Hanaoka §,#,3 and Masahiro Shirakawa †,2 ¶ Center for Priority Areas, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo 192-0397, § Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako 351-0198, † Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo, Kyoto 615-8510, & Ajinomoto Company, Kawasaki, Kawasaki 210-8681, ‡ Division of Structural Biology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki, Chuo, Kobe, Hyogo 650-0017, # Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima, Tokyo 171-8588, Japan. Running title: Structure of the C-terminal region of Cdt1 Keywords: NMR, protein structure, DNA replication initiation, licensing factor, Cdt1 *These authors equally contributed to the work. 1 To whom correspondence may be addressed: Tel.: 81-42-677-4873; Fax: 81-42-677-4873; E-mail: jee-jungoo@tmu.ac.jp 2 To whom correspondence may be addressed: Tel.: 81-75-383-2535; Fax: 81-75-383-2541; E-mail: shirakawa@moleng.kyoto-u.ac.jp 3 To whom correspondence may be addressed: Tel.: 81-3-3986-0221; Fax: 81-3-5992-1029; E-mail: fumio.hanaoka@gakushuin.ac.jp In eukaryotes, DNA replication is fired once in a single cell cycle before cell division starts to maintain stability of the genome. This event is tightly controlled by a series of proteins. Cdt1 is one of the licensing factors and is involved in recruiting replicative DNA helicase Mcm2-7 proteins into the pre-replicative complex together with Cdc6. In Cdt1, the C-terminal region serves as a binding site for Mcm2-7 proteins, although the details of these interactions remain largely unknown. Here, we report the structure of the region and the key residues for binding to Mcm proteins. We determined the solution structure of the C-terminal fragment, residues 450-557, of mouse Cdt1 by NMR. The structure consists of a winged-helix domain and shows unexpected similarity to those of the C-terminal domain of Cdc6 and the central fragment of Cdt1, thereby http://www.jbc.org/cgi/doi/10.1074/jbc.M109.075333 The latest version is at JBC Papers in Press. Published on March 24, 2010 as Manuscript M109.075333 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on February 9, 2016 http://www.jbc.org/ Downloaded from