Schepers A, Diffley J F
Clare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, EN6 3LD, UK.
J Mol Biol. 2001 May 11;308(4):597-608. doi: 10.1006/jmbi.2001.4637.
The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.
Cdc6蛋白是在芽殖酵母DNA复制起点将Mcm2 - 7家族成员复合物（MCM复合物）加载到前复制复合物所必需的。Cdc6p是AAA(+)超家族蛋白的成员，该家族包括原核和真核钳位装载蛋白。这些蛋白共享一些保守的同源区域和共同的三维结构。两个保守的序列基序是沃克A和B基序，它们参与核苷酸代谢，并且对于Cdc6p在体内的功能至关重要。在这里，我们分析了其他保守序列基序中的突变体。其中几个突变体对生长是温度敏感的，并且在限制温度下无法将MCM复合物募集到染色质上。在一个这样的温度敏感突变体中，传感器I基序中一个高度保守的天冬酰胺残基被改变为丙氨酸。这种突变蛋白的过表达是致死的。该表型与先前描述的沃克B基序突变的表型非常相似，表明传感器I和沃克B基序在Cdc6功能中具有共同作用。
