Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism
Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism
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DOI：
10.1093/emboj/19.14.3799
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发表时间：
2000-07-17
期刊：
影响因子：
11.4
通讯作者：
Wigley, DB
中科院分区：
文献类型：
--
作者：
Soultanas, P;Dillingham, MS;Wigley, DB
与 3' 尾 DNA 双链体复合的 PcrA 解旋酶的 DNA 足迹和核酸酶保护研究揭示了在存在和不存在 ATP、ADPNP 的不可水解类似物的情况下覆盖底物的重要区域的接触区域。然而，酶与双链体区域相互作用的细节在核苷酸结合时发生改变。通过将此信息与从与类似 DNA 底物复合的 PcrA 晶体结构中获得的信息相结合，我们设计了解旋酶活性有缺陷但 ATP 酶和单链 DNA 易位活性完整的突变蛋白。这些突变体均位于结构域 1B 和 2B，与 DNA 底物的双链体部分相互作用。结合晶体结构，这些数据支持 PcrA 的“主动”机制，该机制涉及两个不同的 ATP 依赖性过程：在酶之前双链 DNA 的不稳定，该酶与沿单链产物的 DNA 易位偶联。
DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3'-tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non-hydrolysable analogue of ATP, ADPNP, However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single-stranded DNA translocation activities intact, These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an 'active' mechanism for PcrA that involves two distinct ATP-dcpendent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.