A Conservative Isoleucine to Leucine Mutation Causes Major Rearrangements and Cold Sensitivity in KlenTaq1 DNA Polymerase
| Autor: | Wayne M. Barnes, Carol A. Parish, Milko B. Kermekchiev, Bill R. Miller, Erica Modeste, Bryan Zielinski, Amanda R. Walsh, Emma C. Materne, Emily P. Hiltner, Lily Mawby, Eugene Wu |
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| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Base pair DNA polymerase Stereochemistry DNA polymerase II Molecular Dynamics Simulation 010402 general chemistry Crystallography X-Ray 01 natural sciences Biochemistry Article 03 medical and health sciences chemistry.chemical_compound Leucine Taq Polymerase Isoleucine Polymerase 030304 developmental biology 0303 health sciences DNA clamp biology Thermus aquaticus Nucleotides DNA biology.organism_classification 0104 chemical sciences Cold Temperature Kinetics chemistry Mutation biology.protein Mutant Proteins DNA polymerase I Taq polymerase |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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 A 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. |
| Databáze: | OpenAIRE |
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