Comparison of the Incorporation of Watson-Crick Complementary and Mismatched Nucleotides Catalyzed by DNA Polymerase I

Abstract

During the DNA replication and repair synthesis, DNA polymerases add deoxynucleotides onto the growing end of a DNA primer strand using a singlestranded DNA as a template. To maintain the genomic integrity without the expensive proofreading performed by exonucleases, these polymerases have evolved a very high fidelity with error frequencies of approximately one in 103 106 bases synthesized. But for the demands of numerous biotechnical applications even this fidelity is not satisfactory. Especially the unnatural conditions of several techniques either restrict the use of these enzymes or demand their tedious optimization. Thus, the primary design goal for DNA polymerases with altered functions is high specificity in formation of Watson- Crick base pairs during DNA synthesis. To support this research, Summerer et al. [1] presented an efficient automated high-throughput setup for the rapid parallel screening of DNA polymerase mutant libraries. With this technique, they were able to identify several active variants of the Klenow fragment of DNA polymerase I from thermus aquaticus with significant higher extension fidelity than the wild-type enzyme. These variants can now be analyzed concerning structural changes in the ternary complexes composed out of the enzyme, the DNA primer and template as well as the incoming nucleotide with the goal to rationalize the higher specificity.