Dimitrova D S, Todorov I T, Melendy T, Gilbert D M
Department of Biochemistry and Molecular Biology, S.U.N.Y. Health Science Center, Syracuse, New York 13210, USA.
J Cell Biol. 1999 Aug 23;146(4):709-22. doi: 10.1083/jcb.146.4.709.
Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1-mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.
此前在非洲爪蟾卵提取物中进行的实验，鉴定出了似乎是用于DNA复制的两个独立组装的复制前复合体（pre-RC）：ORC、Cdc6和Mcm逐步组装到染色质上，以及FFA-1介导的RPA募集到染色质上的焦点区域。我们研究了在中国仓鼠卵巢（CHO）细胞中是否能检测到这两种pre-RC。用卤化核苷酸对早期和晚期复制的染色体区域进行脉冲标记，并在随后的G1期的不同时间点对预标记的细胞进行同步化处理。研究了Mcm2和RPA募集到这些区域与核膜形成、二氢叶酸还原酶（DHFR）复制起点的确定以及进入S期之间的关系。从末期后期到整个G1期，Mcm2逐渐累积地加载到早期和晚期复制的染色质上。在S期，可检测到的Mcm2迅速从含有PCNA的活跃复制叉中被排除。相比之下，直到S期开始时才能检测到抗去污剂的RPA焦点区域，此时RPA仅与最早启动的复制子结合。在S期，RPA与PCNA特异性地存在于活跃的复制叉处。总之，我们的数据表明，Mcm蛋白而非RPA在G1期早期哺乳动物pre-RC的形成中发挥作用。
