4N5S

Ternary complex structure of Klenow fragment of Taq DNA polymerase I707L mutant (Cs3C KlenTaq) with DNA and ddCTP

Summary for 4N5S

• Entry DOI: 10.2210/pdb4n5s/pdb
• Related: 4N56
• Descriptor: 5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(DOC))-3', 5'-D(*AP*A*AP*GP*GP*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3', DNA polymerase I, thermostable, ... (10 entities in total)
• Functional Keywords: dna polymerase, transferase-dna complex, transferase/dna
• Biological source: Thermus aquaticus
• Total number of polymer chains: 3
• Total formula weight: 72269.94

Authors

Wu, E.Y. (deposition date: 2013-10-10, release date: 2015-01-21, Last modification date: 2023-09-20)

Primary citation

Wu, E.Y.,Walsh, A.R.,Materne, E.C.,Hiltner, E.P.,Zielinski, B.,Miller, B.R.,Mawby, L.,Modeste, E.,Parish, C.A.,Barnes, W.M.,Kermekchiev, M.B.
A Conservative Isoleucine to Leucine Mutation Causes Major Rearrangements and Cold Sensitivity in KlenTaq1 DNA Polymerase.
Biochemistry, 54:881-889, 2015

PubMed 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.

PubMed: 25537790
DOI: 10.1021/bi501198f

Experimental method

X-RAY DIFFRACTION (1.67 Å)