Intrinsic properties of a KlenTaq DNA polymerase mutant with enhanced substrate specificity determined by X-ray structure analysis

The catalytically active Klenow fragment of an evolved Thermus aquaticus DNA polymerase I (KlenTaq) with enhanced substrate specificity was structurally investigated during this Ph.D. thesis. The wild-type KlenTaq is not able to amplify DNA damages like abasic sites or 8-oxo guanine and exhibits no lesion bypass activity. An evolved Thermus aquaticus (Taq) DNA polymerase with an exchange of a hydrophobic methionine to a cationic lysine at position 747 was achieved in the AG Marx and described in detail by C. Gloeckner. This single amino acid mutation led to an enzyme which can process such damaged DNA. Patel et al. found in the KlenTaq a mutation at a different position, I614K, which interestingly exhibits similar properties. This mutant was integrated in the KlenTaq M747K mutant and characterized by C. Gloeckner. This KlenTaq M747K, I614K mutant possessed a better lesion bypass ability combined with a high processivity.The aim of this work in structural biology was to explain the differences between the functional properties of the mutated protein and the wild-type. Therefore, the proteins were crystallized and measured at the synchrotron, either the European Synchrotron Radiation Facility, ESRF, in Grenoble, France, or the Swiss Light Source, SLS, in Villigen, Switzerland.A resolution of up to 1.7 Å was obtained in this work. Until now, this is the highest resolution achieved for the Klenow fragment of the Thermus aquaticus DNA polymerase I. One structure of the KlenTaq in the open, uncomplexed form, three structures in the closed form in complex with primer/template and dideoxynucleotidetriphosphate (ddNTP) and two structures in the closed form in complex with primer/abasic template and ddNTP were solved.The structures reveal, that the higher positive charge of the polymerase at position 747 obviously results in a tighter binding of the DNA. This stronger positive potential is also observed in the translesion bypass polymerases (e.g. DPO4 - polymerase), whereas the family I DNA polymerases exhibit a less stronger positive potential in the DNA binding region. Moreover, a hydrogen bond network could be observed, which is absent in the wild-type. This also seems to contribute to a tighter binding of the protein to the template.Interestingly, in the structure of the KlenTaq M747K, I614K in complex with p/t and ddATP there is again a nonpolar to cationic exchange from isoleucine to lysine at position 614.Lysine 614 interacts with a hydrogen bond network with motif C and motif C is responsible for the binding of the metal ions, which are responsible for the incorporation of the triphosphate and for the dephosphorylation step. This interaction seems to trap the binding pocket in a more open conformation. In the KlenTaq M747K, I614K structure, bound to a stabilized abasic site template, this hydrogen bond network was visible, too, and the phenyl ring of tyrosine 671 is in the position of the absent coding base of the template. Opposite this tyrosine, an adenosintriphosphate is bound to the incorporation site via a hydrogen bond and base stacks between the adjacent nucleobase of the 3'- end of the primer and F667 from the protein. This is the first time, that such an incorporation opposite an abasic site was ever observed in a crystal structure. Soaking experiments with ddGTP instead of the bound ddATP led to a structure, where the guanosine is incorporated opposite the abasic site. The incorporated guanosine is hydrogen bonded to glutamate 615, while the adenosine hydrogen bonds to aspartate 785. The ten times higher rate of incorporation of adenosine compared to guanosine may be due to intrinsic properties of these purines. The better solvation of adenosine together with the lower hydration energy and the superior base stacking explains the higher incorporation rate compared with guanosine. Moreover, replicative polymerases enclose at the 3'- end of the DNA primer adenosine overhangs following the A-rule. The A-rule can also be explained by the tyrosine mimicking pyrimidine