Dillingham M S, Soultanas P, Wiley P, Webb M R, Wigley D B
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8381-7. doi: 10.1073/pnas.131009598.
Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues. We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity. We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA. Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.
PcrA解旋酶的晶体结构和生化分析为一种连续DNA解旋模型提供了证据，该模型涉及单链DNA（ssDNA）追踪与双链去稳定化活性的耦合。DNA追踪模型引发了由保守芳香族氨基酸残基形成的酶上几个口袋之间碱基的ATP依赖性翻转。我们已使用定点诱变来确认所有这些残基对解旋酶活性的必要性。我们还证明，双链解旋缺陷与某些突变蛋白无法在ssDNA上有效转运有关。此外，结果确定了ssDNA结合位点内一个必需的三联体残基，其构成了ATP驱动的DNA马达本身。
