Okada Takuya, Okabe Gaku, Tak Yon-Soo, Mimura Satoru, Takisawa Haruhiko, Kubota Yumiko
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto, Japan.
Genes Cells. 2018 Feb;23(2):94-104. doi: 10.1111/gtc.12556. Epub 2018 Jan 4.
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.
从静止细胞中分离出的完整G0期细胞核在非洲爪蟾卵提取物的间期不能进行DNA复制，而这种提取物能使通透的G0期细胞核高效复制。先前的研究已经表明存在多种维持静止状态的调控机制，但对于提取物中完整G0期细胞核的DNA复制抑制作用仍知之甚少。在此，我们表明前复制复合体（pre-RC）在染色质上组装，但其激活在将从静止的NIH3T3细胞中分离出的G0期细胞核在提取物中孵育后受到抑制。伴随着复制的抑制，在G0期细胞核中，由Dbf4依赖性激酶（DDK）介导的Mcm4磷酸化以及DDK与染色质的结合受到抑制，而不影响DDK的核转运。我们进一步发现，G0期而非增殖细胞的核提取物抑制重组DDK与组装了pre-RC的质粒的结合。此外，我们观察到将G0期细胞核在卵提取物中孵育后检查点激酶迅速激活。然而，ATR/ATM的特异性抑制剂不能促进卵提取物中G0期细胞核的DNA复制。我们认为一种新的抑制机制发挥作用，以阻止DDK靶向G0期细胞核中的pre-RC，从而抑制非洲爪蟾卵提取物中的DNA复制。
