Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4
Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4
复制标题
人类 MCM-BP 蛋白与 MCM 复合物成分和 Dbf4 的相互作用
作者：
T. Nguyen;M. Jagannathan;K. Shire;L. Frappier
MCM-BP被发现是一种从人类细胞中与MCM蛋白3至7共纯化的蛋白质;结果在青蛙、酵母和植物中重现。所有这些生物体中的证据支持MCM-BP在DNA复制中的重要作用，包括对MCM复合物卸载的贡献。然而，MCM-BP功能和与MCM复合物缔合的机制还不清楚。在这里，我们表明，人类MCM-BP是能够相互作用的单个MCM蛋白2至7时，在昆虫细胞中共表达，并可以大大增加一些重组MCM蛋白的回收率。甘油梯度沉降分析表明，MCM-BP与MCM 4和MCM 7的相互作用最强。人细胞裂解物的类似梯度分析表明，只有少量的MCM-BP与MCM复合物的迁移重叠，并且MCM复合物被外源性MCM-BP破坏。此外，在S期中后期检测到含有MCM-BP和MCM蛋白的大复合物，表明特异性MCM-BP复合物的形成受细胞周期调节。我们还确定了MCM-BP之间的相互作用和Dbf 4的DDK激酶的调节组分在酵母双杂交和昆虫细胞共表达试验，这种相互作用的内源性蛋白质从人类细胞的免疫共沉淀验证。体外激酶试验表明，MCM-BP不是DDK的底物，但可以抑制MCM 4，6，7或MCM 2 -7复合物中MCM 4，6，7的DDK磷酸化，对MCM 2的DDK磷酸化几乎没有影响。由于DDK是已知的激活DNA复制通过这些MCM蛋白的磷酸化，我们的研究结果表明，MCM-BP可能会影响DNA复制的一部分，通过调节MCM磷酸化DDK。
MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.
登录
查看更多内容
影响因子：
16
作者：
Chuang, Li-Chiou;Teixeira, Leonardo K.;Wohlschlegel, James A.;Henze, Martha;Yates, John R.;Mendez, Juan;Reed, Steven I.
通讯作者：
Reed, Steven I.
影响因子：
16
作者：
Hubbi ME;Luo W;Baek JH;Semenza GL
通讯作者：
Semenza GL
通过与CDC45和杜松子蛋白缔合对MCM2-7解旋酶的激活。
影响因子：
16
作者：
Ilves I;Petojevic T;Pesavento JJ;Botchan MR
通讯作者：
Botchan MR
影响因子：
16
作者：
Sheu, Yi-Jun;Stillman, Bruce
通讯作者：
Stillman, Bruce
影响因子：
16
作者：
Tsuji T;Lau E;Chiang GG;Jiang W
通讯作者：
Jiang W