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Vol. 20, Issue 3, 937-947, February 1, 2009

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Repression of Nascent Strand Elongation by Deregulated Cdt1 during DNA Replication in Xenopus Egg Extracts

Takashi Tsuyama^*, Saori Watanabe^*, Ayako Aoki^*, Yunje Cho, Masayuki Seki^*, Takemi Enomoto^*,, and Shusuke Tada^*

*Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, 980-8578, Japan; National Creative Research Center for Structural Biology and Department of Life Science, Pohang University of Science and Technology, Pohang, 790-784, South Korea; and Tohoku University 21st Century COE Program, "Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation," Sendai, Miyagi, 980-8578, Japan

Submitted June 16, 2008; Revised November 18, 2008; Accepted November 24, 2008
Monitoring Editor: Orna Cohen-Fix

Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system.

Address correspondence to: Shusuke Tada (tada{at}mail.pharm.tohoku.ac.jp)