Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts.
Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.
Tsuyama
Takashi
T
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Tada
Shusuke
S
Watanabe
Saori
S
Seki
Masayuki
M
Enomoto
Takemi
T
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002457:Cell Extracts; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism*
C118388:Cdc6 protein, Xenopus; C406819:Cdt1 protein, Xenopus; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D029867:Xenopus Proteins