Shechter David, Ying Carol Y, Gautier Jean
Integrated Program in Cellular, Molecular, and Biophysical Studies, Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
J Biol Chem. 2004 Oct 29;279(44):45586-93. doi: 10.1074/jbc.M407772200. Epub 2004 Aug 23.
Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activities in vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) from Xenopus laevis eggs and six purified polyclonal antibodies generated against each of the Xenopus Mcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2-7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21(cip1) specifically blocks origin firing but does not prevent helicase action. When p21(cip1) is added, followed by the non-hydrolyzable analog ATPgammaS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.
微小染色体维持蛋白（Mcm）在所有真核生物中都是必不可少的，并且是DNA复制起始所绝对必需的。真核生物和古细菌的Mcm蛋白具有保守的解旋酶基序，并且在体外表现出DNA解旋酶和ATP水解活性。尽管有人提出Mcm蛋白是复制性解旋酶，即在复制叉处解开DNA螺旋的酶，但其在细胞DNA复制延伸过程中的功能仍不清楚。利用非洲爪蟾卵的核质提取物（NPE）和针对每种非洲爪蟾Mcm蛋白产生的六种纯化多克隆抗体，我们已经证明Mcm蛋白在DNA复制期间以及复制起始后的DNA解旋过程中是必需的。从NPE中定量去除Mcm会导致正常的复制和解旋，这证实了Mcm在复制前复合体组装之前是必需的，而在那之后则是可有可无的。当在NPE孵育期间加入针对六种不同Mcm2 - 7蛋白的混合中和抗体时，复制和解旋受到抑制。此外，在NPE中经过初始复制期后加入Mcm抗体可阻断复制，这通过在加入抗体的同时脉冲标记放射性核苷酸来可视化。加入细胞周期蛋白依赖性激酶2抑制剂p21（cip1）可特异性阻断起始点激发，但不阻止解旋酶作用。当加入p21（cip1），随后加入不可水解的类似物ATPγS以阻断解旋酶功能时，解旋受到抑制，这表明质粒解旋特别归因于ATP水解依赖性功能。这些数据支持了Mcm蛋白复合体作为复制性解旋酶发挥作用的假说。
