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Suppression of targeting of Dbf4‐dependent kinase to pre‐replicative complex in G0 nuclei
Author(s) -
Okada Takuya,
Okabe Gaku,
Tak YonSoo,
Mimura Satoru,
Takisawa Haruhiko,
Kubota Yumiko
Publication year - 2018
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12556
Subject(s) - biology , xenopus , dna replication , chromatin , microbiology and biotechnology , kinase , cell cycle , dna , interphase , cell , biochemistry , gene
Intact G0 nuclei isolated from quiescent cells are not capable of DNA replication in interphase Xenopus egg extracts, which allow efficient replication of permeabilized G0 nuclei. Previous studies have shown multiple control mechanisms for maintaining the quiescent state, but DNA replication inhibition of intact G0 nuclei in the extracts remains poorly understood. Here, we showed that pre‐RC is assembled on chromatin, but its activation is inhibited after incubating G0 nuclei isolated from quiescent NIH3T3 cells in the extracts. Concomitant with the inhibition of replication, Mcm4 phosphorylation mediated by Dbf4‐dependent kinase (DDK) as well as chromatin binding of DDK is suppressed in G0 nuclei without affecting the nuclear transport of DDK. We further found that the nuclear extracts of G0 but not proliferating cells inhibit the binding of recombinant DDK to pre‐RC assembled plasmids. In addition, we observed rapid activation of checkpoint kinases after incubating G0 nuclei in the egg extracts. However, specific inhibitors of ATR/ATM are unable to promote DNA replication in G0 nuclei in the egg extracts. We suggest that a novel inhibitory mechanism is functional to prevent the targeting of DDK to pre‐RC in G0 nuclei, thereby suppressing DNA replication in Xenopus egg extracts.