Weinreich M, Liang C, Stillman B
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):441-6. doi: 10.1073/pnas.96.2.441.
Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin-associated factor. It then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologues contain a bipartite Walker ATP-binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The Cdc6(K114E) protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase. These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.
Cdc6p在DNA复制起始的机制和调控中具有重要作用。芽殖酵母Cdc6p在M期末期至G1期早期通过与起源识别复合物或其他与起源相关的因子相互作用，结合到自主复制序列元件附近的染色质上。然后，它通过一种未知机制促进Mcm蛋白家族随后在自主复制序列元件附近的加载。所有Cdc6p同源物都包含一个双分型沃克ATP结合基序，这表明ATP结合或水解可能调节Cdc6p的活性。为了确定这些基序对Cdc6p活性是否重要，在沃克A和B基序的保守残基中进行了突变。将沃克A基序中的赖氨酸114替换为丙氨酸（K114A），在酵母中导致温度敏感型表型，并且在允许温度下进入S期的进程变慢。K114E突变是致死的。Cdc6(K114E)蛋白能结合到染色质上，但不能促进Mcm蛋白的加载，这表明ATP结合对于该活性至关重要。与Cdc6p缺失不同，该突变体停滞在G1期DNA含量状态，但在没有DNA复制的情况下仍保留抑制有丝分裂的能力。相比之下，在沃克B基序中含有双丙氨酸突变DE(223, 224)AA的Cdc6p是有功能的，并且该突变体表现出明显正常的S期。这些结果表明，Cdc6p核苷酸结合对于在DNA复制起点建立复制前复合物很重要，并且Cdc6p的氨基末端对于阻止进入有丝分裂是必需的。
