Structure of Taq polymerase with DNA at the polymerase active site

Abstract

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^1,2. The structures of 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 enzyme². The structures have been solved for editing complexes between KF and single-stranded DNA^3,4 and for duplex DNA with a 3′ overhanging single strand⁵, 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 ångströms along their helix axes.