Reconstitution of the Mcm2-7p Heterohexamer, subunit arrangement, and ATP site architecture
Reconstitution of the Mcm2-7p Heterohexamer, subunit arrangement, and ATP site architecture
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DOI：
10.1074/jbc.m210511200
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发表时间：
2003-02-14
影响因子：
4.8
通讯作者：
O'Donnell, M
中科院分区：
文献类型：
--
作者：
Davey, MJ;Indiani, C;O'Donnell, M
Mcm 2 - 7 p异六聚体是真核细胞中假定的复制解旋酶。这六个亚单位中的每一个都是复制所必需的。我们已经纯化了6个酿酒酵母MCM蛋白作为重组蛋白在大肠杆菌中，并已重建的Mcm 2 - 7 p复合物从个别亚基。MCM ATP酶活性研究表明，MCM蛋白不能有效地水解ATP。ATP水解需要两种MCM蛋白的组合。MCM蛋白质的15种可能的成对混合物仅产生3对产生ATP酶活性的MCM蛋白质。对Mcm 3/7 p ATP酶的研究表明，Mcm 3 p中的必需精氨酸是水解与Mcm 7 p结合的ATP所必需的。MCM蛋白之间的成对相互作用的研究将剩余的MCM蛋白与Mcm 3/7 p对连接。这些数据预测了ATP酶对中的哪些亚基结合水解的ATP，并指示了Mcm 2 - 7 p异六聚体中亚基的排列。
The Mcm2-7p heterohexamer is the presumed replicative helicase in eukaryotic cells. Each of the six subunits is required for replication. We have purified the six Saccharomyces cerevisiae MCM proteins as recombinant proteins in Escherichia coli and have reconstituted the Mcm2-7p complex from individual subunits. Study of MCM ATPase activity demonstrates that no MCM protein hydrolyzes ATP efficiently. ATP hydrolysis requires a combination of two MCM proteins. The fifteen possible pairwise mixtures of MCM proteins yield only three pairs of MCM proteins that produce ATPase activity. Study of the Mcm3/7p ATPase shows that an essential arginine in Mcm3p is required for hydrolysis of the ATP bound to Mcm7p. Study of the pairwise interactions between MCM proteins connects the remaining MCM proteins to the Mcm3/7p pair. The data predict which subunits in the ATPase pairs bind the ATP that is hydrolyzed and indicate the arrangement of subunits in the Mcm2-7p heterohexamer.