Repression of nascent strand elongation by deregulated Cdt1 during DNA replication in Xenopus egg extracts.
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.
Tsuyama
Takashi
T
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, 980-8578, Japan.
Watanabe
Saori
S
Aoki
Ayako
A
Cho
Yunje
Y
Seki
Masayuki
M
Enomoto
Takemi
T
Tada
Shusuke
S
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002457:Cell Extracts; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D064248:Geminin; D009154:Mutation / Q000235:genetics; D010063:Ovum / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism*
C406819:Cdt1 protein, Xenopus; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; C577998:GMNN protein, Xenopus; D064248:Geminin; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D002110:Caffeine