Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity
Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity
复制标题
DOI：
10.1074/jbc.m206202200
复制
发表时间：
2002-10-25
影响因子：
4.8
通讯作者：
Hanaoka, F
中科院分区：
文献类型：
--
作者：
Yanagi, K;Mizuno, T;Hanaoka, F
DNA复制由复制前复合体和复制装置的逐步组装控制。CDT1是复制前复合体的一种新成分，在将微小染色体维持(MCM)2-7复合体装载到染色质上起着重要作用。在后生动物细胞的G2/M转变过程中，cdt1活性受Geminin的抑制。为了了解哺乳动物细胞中cdt1-ginin调控机制的分子基础，我们克隆并在细菌细胞中表达了小鼠cdt1同源基因，并将小鼠cdt1纯化到接近同源性。我们通过酵母双杂交分析发现，小鼠CDT1与Ginin、MCM6和起源识别复合体2结合。MCM6与真核生物中保守的CDT1羧基末端区域(氨基酸407-477)相互作用，而Gminin与只有后生动物保守的CDT1中心区(氨基酸177-380)结合。此外，我们还发现，CDT1可以以序列、链和构象无关的方式与DNA结合。CDt1的DNA结合域与Ginin结合域重叠，Ginin抑制了CDt1与DNA的结合。综上所述，我们定义了小鼠CDT1的结构域和新的生化性质，表明CDT1在复制前复合体中作为一种内在的DNA结合因子发挥作用。
DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G2/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.