Allen G C, Kornberg A
Department of Biochemistry, Stanford University School of Medicine, California 94305.
J Biol Chem. 1991 Nov 25;266(33):22096-101.
The DnaC protein of Escherichia coli is essential for replication in vivo and in vitro. In the initiation of replication of a minichromosome at its origin, DnaC delivers the DnaB helicase from a DnaB.DnaC complex to the future replication fork and then departs. However, if an excess of DnaC was present in subsequent steps, it severely inhibited replication by slowing the DnaB helicase at the replication fork. When DnaB was present at a level equimolar with the excess DnaC, the inhibition was relieved, implying that the ratio of DnaC to DnaB is critical for achieving optimal replication activity and avoiding inhibition by DnaC. In vivo, overproduction of DnaC slowed cell growth. This slowing was alleviated by overproducing DnaB at the same time. E. coli strains with a dnaCts gene defective in chromosomal initiation were complemented by the wild-type gene in trans. On the other hand, strains with an elongation-defective dnaCts gene were not complemented by the wild-type dnaC gene. The dominance of the mutant protein suggests that it remains tightly complexed with DnaB at the replication fork, inhibiting elongation even in the presence of the wild-type DnaC.
大肠杆菌的DnaC蛋白对于体内和体外复制都是必需的。在微型染色体于其起始点开始复制时，DnaC将DnaB解旋酶从DnaB.DnaC复合物传递至未来的复制叉，然后离开。然而，如果在后续步骤中存在过量的DnaC，它会通过减缓复制叉处的DnaB解旋酶而严重抑制复制。当DnaB的水平与过量的DnaC等摩尔时，抑制作用得以缓解，这意味着DnaC与DnaB的比例对于实现最佳复制活性和避免被DnaC抑制至关重要。在体内，DnaC的过量表达会减缓细胞生长。同时过量表达DnaB可缓解这种生长减缓。具有在染色体起始方面有缺陷的dnaCts基因的大肠杆菌菌株可被野生型基因反式互补。另一方面，具有延伸缺陷型dnaCts基因的菌株不能被野生型dnaC基因互补。突变蛋白的显性表明它在复制叉处与DnaB紧密结合，即使在存在野生型DnaC的情况下也会抑制延伸。
