Date of Award
2014
Document Type
Thesis
Degree Name
Bachelor of Science
Department
Biochemistry & Molecular Biol.
First Advisor
Dr. Eugene Wu
Second Advisor
Dr. Michelle Hamm
Abstract
Polymerase Chain Reaction (PCR) typically employs the Taq or Klentaq1 DNA polymerase from Thermus aquaticus to elongate short sequences of DNA during DNA amplification. Both Taq and Klentaq1 retain activity at room temperature. During assembly of PCR at room temperature, the activity of Taq and Klentaq1 can result in spurious products due to elongation of primers bound to non-target sequences. A mutation of isoleucine to leucine at position 707 of Klentaq1 results in slowing of the enzyme at room temperature without compromising the fidelity of the enzyme. To understand how a mutation over 20Å from the active site can affect the performance of an enzyme, we solved the X-ray crystal structure of the ternary (E-DNA-ddNTP) and binary (E-DNA) complexes of the Klentaq1 polymerase with the point mutation I707L (Cs3C). The Cs3C ternary complex showed the mutation in conjunction with a rotameric change of a nearby phenylalanine 749. The Cs3C mutant structure was otherwise identical to the Klentaq1 wild-type, suggesting that it remained active. The solved binary structure resulting from soaking out the ddCTP of the ternary complex again showed a rotameric change of the phenylalanine 749. This rotameric change resulted in a vacated space in the P-helix and widening of the cleft between the hand and palm motifs, allowing two adenosines in the DNA template strand to stack in the active site. This base-stacking blocked the active site, thereby slowing the enzyme during nucleotide incorporation and impeding the transition from open to closed conformations. Replacement of the adenosines with thymidines resulted in an open, unblocked conformation, confirming the importance of base-stacking during nucleotide addition. These results explain the cold-sensitive phenotype observed in the Cs3C mutant and the influence of a distant point mutation on conformational change.
Recommended Citation
Materne, Emma Caroline, "A conservative isoleucine to leucine mutation of Klentaq1 dna polymerase 1 induces conformational change for cold-sensitive phenotype" (2014). Honors Theses. 877.
https://scholarship.richmond.edu/honors-theses/877