A Conservative Isoleucine to Leucine Mutation Causes Major Rearrangements and Cold Sensitivity in KlenTaq1 DNA Polymerase

Abstract

Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary (E–DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5′-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.

Combinatorial Genetic Modeling ofpfcrt-Mediated Drug Resistance Evolution inPlasmodium falciparum

“…In agreement with both studies, we observed Ecu A K76T to be insufficient for CQR and uncovered direct contributions to CQR by additional PfCRT mutations, in particular Ecu C N326D. Crystallographic insights, presently lacking for PfCRT or its homologs, indicate that even seemingly conservative substitutions (e.g., isoleucine to leucine, as in I356L) can significantly impact a protein’s structure and function ( Wu et al 2015 ). In addition to PfCRT I356L, the apparent need for mutational acquisition of basic, polar, and acidic side chains at residues 76, 220, and 326, respectively, suggests specific electrostatic and conformational requirements for PfCRT-mediated CQR.…”

Nuclear trafficking of the anti-apoptoticCoxiella burnetiieffector protein AnkG requires binding to p32 and Importin-α1

“…Interestingly, this is not the only case of a radical alteration caused by isoleucine to leucine replacement. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity (Wu et al, 2015). For AnkG-mediated activity the consequences are only subtle.…”

Structural basis for the selective incorporation of an artificial nucleotide opposite a DNA adduct by a DNA polymerase

“…Ternary DNA polymerase complexes with manganese ions have already been obtained before and the position as well as the coordination patterns of the ions in the active sites were highly similar compared to the smaller magnesium ions. 19,20 The M747K G:C structure was solved by cocrystallization of KlenTaq M747K with a natural primer/template complex and dCTP in the presence of magnesium ions. It is very similar to the KlenTaq WT structure with the same primer/template and substrate complexed (PDB ID.…”