Studien zum Substratspektrum von DNA-Polymerasen

Abstract

An essential prerequisite for all organisms is to keep its genome intact and to accurately duplicate it prior to cell division. All DNA synthesis is catalyzed by DNA polymerases, which play a crucially role in the accuracy of informationtransfer. The fidelity of DNA replication depends on the ability of DNA polymerases to recognize the template and insert the accurat nucleotide according to Watson-Crick basepairing.During DNA replication not only the incorporation of the accurat nucleobase, but also the configuration of the ribose unit seems to be regulated, as natural DNA and RNA is exclusively composed of D-enantiomers. So far it has not been understood how nature has chosen and retained chirality of DNA biosynthesis during evolution. The differentiation between the two stereoisomers requires specific contacts of the DNA polymerases with the sugar residues of the substrates in addition to the interactions with the nucleobase, which ensure selective formation of Watson-Crick basepairs.The aim of this work was to evolve a DNA polymerase with the ability to incorporate L-nucleotides.We designed preliminary experiments, testing several commercially available DNA polymerases from different distinct DNA polymerase families for their ability to incorporate L-thymidinetriphosphate (L-TTP). Out of those DNA polymerases, the Therminator DNA polymerase stood out due to its ability to catalyze the insertion of two L-TTP. This property appears to be related to a mutation in its finger domain.Based on these results, DNA polymerases with improved properties should have been generated using the directed evolution approach. Therefore, two evolution systems were used.