Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2-7 complex.
Mcm2-7 is recruited to eukaryotic origins of DNA replication by origin recognition complex, Cdc6 and Cdt1 thereby licensing the origins. Cdc6 is essential for origin licensing during DNA replication and is readily destabilized from chromatin after Mcm2-7 loading. Here, we show that after origin licensing, deregulation of Cdc6 suppresses DNA replication in Xenopus egg extracts without the involvement of ATM/ATR-dependent checkpoint pathways. DNA replication is arrested specifically after chromatin binding of Cdc7, but before Cdk2-dependent pathways and deregulating Cdc6 after this step does not impair activation of origin firing or elongation. Detailed analyses revealed that Cdc6 deregulation leads to strong suppression of Cdc7-mediated hyperphosphorylation of Mcm4 and subsequent chromatin loading of Cdc45, Sld5 and DNA polymerase α. Mcm2 phosphorylation is also repressed although to a lesser extent. Remarkably, Cdc6 itself does not directly inhibit Cdc7 kinase activity towards Mcm2-4-6-7 in purified systems, rather modulates Mcm2-7 phosphorylation on chromatin context. Taken together, we propose that Cdc6 on chromatin acts as a modulator of Cdc7-mediated phosphorylation of Mcm2-7, and thus destabilization of Cdc6 from chromatin after licensing is a key event ensuring proper transition to the initiation of DNA replication.
Kundu
Lena R
LR
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.
Kumata
Yuji
Y
Kakusho
Naoko
N
Watanabe
Saori
S
Furukohri
Asako
A
Waga
Shou
S
Seki
Masayuki
M
Masai
Hisao
H
Enomoto
Takemi
T
Tada
Shusuke
S
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002843:Chromatin / Q000201:enzymology; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D064111:Minichromosome Maintenance Complex Component 2; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism; D010063:Ovum / Q000201:enzymology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis
C118388:Cdc6 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; C576898:Atr protein, Xenopus; C068619:CDC7 protein, human; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D051357:Cyclin-Dependent Kinase 2; D004265:DNA Helicases; C516793:Mcm4 protein, mouse; C491205:Mcm6 protein, mouse; C491086:Mcm7 protein, mouse; D064111:Minichromosome Maintenance Complex Component 2; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7