The role of I614 and E615 in unnatural substrate recognition in Taq DNA polymerase mutants (967.5)

Abstract

DNA polymerases enable key DNA biotechnologies by performing enzymatic amplification of DNA with high efficiency and fidelity, enabling DNA's use in polymerase chain reaction (PCR), sequencing techniques and many different applications in therapeutics and diagnostics. While these astounding properties of DNA polymerases enable the use of these technologies, they also restrict them because they do not recognize modified substrates. Three previous studies have evolved DNA pol I from Thermus aquaticus (Taq) to recognize 2’ modifications of DNA; while these have resulted in an increase in modified substrate recognition, these enzymes are not active enough for practical use. Here, we describe efforts to identify the key mutations that impart unnatural activity on these previously identified enzymes. In particular, we have focused on mutations at I614 and E615 since they occur in all three previously identified mutants. Data show that much of the activity that is present in the previously evolved enzymes is largely a result of the E615G mutation. Mutations at I614 have been found to have no effect when present alone, but have also been found to have varying effects on enzymatic activity when in conjunction with the E615 mutation. Current work is also being done to further investigate the synergistic interactions of mutations of I614 and E615G. These efforts will elucidate key features of the evolution of unnatural substrate recognition by mutant Taq enzymes and will aid in future polymerase engineering efforts.