Eom S H, Wang J, Steitz T A
Department of Molecular Biophysics, Yale University, New Haven, Connecticut 06520-811, USA.
Nature. 1996 Jul 18;382(6588):278-81. doi: 10.1038/382278a0.
The DNA polymerase from Thermus aquaticus (Taq polymerase) is homologous to Escherichia coli DNA polymerase I (Pol I) and likewise has domains responsible for DNA polymerase and 5' nuclease activities. The structures to the polymerase domains of Taq polymerase and of the Klenow fragment (KF) of Pol I are almost identical, whereas the structure of a vestigial editing 3'-5' exonuclease domain of Taq polymerase that lies between the other two domains is dramatically altered, resulting in the absence of this activity in the thermostable enzyme. The structures have been solved for editing complexes between KF and single-stranded DNA and for duplex DNA with a 3' overhanging single strand, but not for a complex containing duplex DNA at the polymerase active-site. Here we present the co-crystal structure of Taq polymerase with a blunt-ended duplex DNA bound to the polymerase active-site cleft; the DNA neither bends nor goes through the large polymerase cleft, and the structural form of the bound DNA is between the B and A forms. A wide minor groove allows access to protein side chains that hydrogen-bond to the N3 of purines and the O2 of pyrimidines at the blunt-end terminus. Part of the DNA bound to the polymerase site shares a common binding site with DNA bound to the exonuclease site, but they are translated relative to each other by several angstroms along their helix axes.
嗜热水生栖热菌的DNA聚合酶（Taq聚合酶）与大肠杆菌DNA聚合酶I（Pol I）同源，同样具有负责DNA聚合酶和5'核酸酶活性的结构域。Taq聚合酶的聚合酶结构域与Pol I的Klenow片段（KF）的结构几乎相同，而位于其他两个结构域之间的Taq聚合酶残余编辑3'-5'外切核酸酶结构域的结构发生了显著改变，导致这种热稳定酶缺乏该活性。已经解析了KF与单链DNA之间的编辑复合物以及具有3'突出单链的双链DNA的结构，但尚未解析在聚合酶活性位点包含双链DNA的复合物的结构。在此，我们展示了Taq聚合酶与结合在聚合酶活性位点裂隙处的平端双链DNA的共晶体结构；DNA既不弯曲也不穿过大的聚合酶裂隙，结合的DNA的结构形式介于B型和A型之间。宽阔的小沟允许蛋白质侧链接近，这些侧链与平端末端嘌呤的N3和嘧啶的O2形成氢键。与聚合酶位点结合的部分DNA与与外切核酸酶位点结合的DNA共享一个共同的结合位点，但它们沿其螺旋轴相对于彼此平移了几埃。
