Mammalian nuclei become licensed for DNA replication during late telophase.
Mammalian nuclei become licensed for DNA replication during late telophase.
复制标题
DOI：
--
复制
发表时间：
2002
影响因子：
4
通讯作者：
D. Dimitrova;T. Prokhorova;J. Blow;I. Todorov;D. Gilbert
中科院分区：
文献类型：
--
作者：
D. Dimitrova;T. Prokhorova;J. Blow;I. Todorov;D. Gilbert
mcm2 -7是哺乳动物细胞核末期与染色质结合的重要复制蛋白。在这里，我们研究了Mcm结合、染色质复制许可和二氢叶酸还原酶（DHFR）复制起源之间的关系。在中国仓鼠卵巢（CHO）细胞中，大约20%的Mcm3蛋白在末期与染色质结合，而另外25%的Mcm3蛋白在g1期逐渐累积结合。为了研究这种结合的功能意义，从中期后不同时间同步的CHO细胞制备的细胞核被引入爪蟾卵提取物中，这些提取物要么是Mcm蛋白的免疫缺失，要么是补充了geminin （Mcm加载蛋白Cdt1的抑制剂）。中期后1小时内，随着核膜形成的完成，CHO细胞核在这两种有缺陷的提取物中完全能够复制。然而，在这些提取物中发现，在中期后1至5小时分离的细胞核中，每个提取物的复制起始位点分散在DHFR位点上，但在中期后5小时分离的细胞核中，它们集中在DHFR原点上。重要的是，将通透性后odp而非前odp的CHO细胞核引入到缺乏许可的爪蟾卵提取物中，导致DHFR起源特异性得到了很大程度的保留，这意味着先前记录的通透性细胞核缺乏特异性起源选择至少部分是由于爪蟾卵提取物中的蛋白质许可了新的起始位点。我们得出结论，在CHO细胞中，Mcm蛋白与染色质的功能性结合（即复制许可）发生在末期，在DHFR位点指定复制起点之前几个小时。
Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.