Bird L E, Brannigan J A, Subramanya H S, Wigley D B
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Nucleic Acids Res. 1998 Jun 1;26(11):2686-93. doi: 10.1093/nar/26.11.2686.
PcrA from Bacillus stearothermophilus is a DNA helicase for which, despite the availability of a crystal structure, there is very little biochemical information. We show that the enzyme has a broad nucleotide specificity, even being able to hydrolyse ethenonucleotides, and is able to couple the hydrolysis to unwinding of DNA substrates. In common with the Escherichia coli helicases Rep and UvrD, PcrA is a 3'-5' helicase but at high protein concentrations it can also displace a substrate with a 5' tail. However, in contrast to Rep and UvrD, we do not see any evidence for dimerisation of the protein even in the presence of DNA. The enzyme shows a specificity for the DNA substrate in gel mobility assays, with the preferred substrate being one with both single and double stranded regions of DNA. We propose that these data, together with existing structural evidence, support an inchworm rather than a rolling model for 3'-5' helicase activity.
嗜热脂肪芽孢杆菌的PcrA是一种DNA解旋酶，尽管已有其晶体结构，但关于它的生化信息却非常少。我们发现该酶具有广泛的核苷酸特异性，甚至能够水解乙烯核苷酸，并且能够将水解作用与DNA底物的解旋相偶联。与大肠杆菌解旋酶Rep和UvrD一样，PcrA是一种3'-5'解旋酶，但在高蛋白浓度下，它也能够置换具有5'尾的底物。然而，与Rep和UvrD不同的是，即使在有DNA存在的情况下，我们也没有发现该蛋白二聚化的任何证据。在凝胶迁移试验中，该酶对DNA底物表现出特异性，其首选底物是同时具有单链和双链DNA区域的底物。我们认为，这些数据与现有的结构证据一起，支持了3'-5'解旋酶活性的尺蠖模型而非滚动模型。
