title: Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts.
abstract: Cdt1 is a protein essential for initiation of DNA replication; it recruits MCM helicase, a core component of the replicative DNA helicase, onto replication origins. In our previous study, we showed that addition of excess Cdt1 inhibits nascent strand elongation during DNA replication in Xenopus egg extracts. In the present study, we investigated the mechanism behind the inhibitory effect of Cdt1. We found that addition of recombinant Cdt1 inhibited nascent DNA synthesis in a reinitiation-independent manner. To identify the mechanism by which Cdt1 inhibits nascent strand elongation, the effect of Cdt1 on loading of Mcm4 and Rpa70 onto chromatin was examined. The results showed that Cdt1 suppressed the excessive Rpa70 binding caused by extensive, aphidicolin-induced DNA unwinding; this unwinding occurs between stalled DNA polymerases and advancing replication forks. These findings suggested that excess Cdt1 suppressed the progression of replication forks.
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Nakazaki
authors.affiliation: Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsuyama
authors.affiliation: Department of Biochemistry, Tohoku Pharmaceutical University, Sendai, Miyagi, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Seki
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo, Japan.
authors.forename: Mikiko
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Nishitokyo-shi, Tokyo, Japan.
authors.forename: Takemi
authors.initials: T
authors.lastname: Enomoto
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo, Japan; Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba, Japan. Electronic address: s.tada@phar.toho-u.ac.jp.
authors.forename: Shusuke
authors.initials: S
authors.lastname: Tada
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D010063:Ovum / Q000502:physiology*; D061805:Transcription Elongation, Genetic / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: C467199:CDT1 protein, human; D018797:Cell Cycle Proteins
keywords: Cdt1; DNA replication; MCM helicase; Xenopus egg extracts