PDBsum entry 4xiu

Transferase/DNA

PDB id: 4xiu

Contents

Protein chain

539 a.a.

DNA/RNA

Metals

_MG

Waters ×1

PDB id:

4xiu

Name:

Transferase/DNA

Title:

Binary complex structure of klenow fragment of taq DNA polymerase i707l mutant with DNA containing ttt overhang

Structure:

Synthetic oligonucleotide primer strand. Chain: b. Engineered: yes. Synthetic oligonucleotide template strand. Chain: c. Engineered: yes. DNA polymerase i, thermostable. Chain: a. Fragment: klenow fragment of DNA polymerase i from thermus aquatics.

Source:

Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Other_details: synthetic oligonucleotide. Thermus aquaticus. Organism_taxid: 271. Gene: pola, pol1. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.50Å R-factor: 0.226 R-free: 0.286

Authors:

E.Y.Wu

Key ref:

E.Y.Wu et al. (2015). A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase. Biochemistry, 54, 881-889. PubMed id: 25537790 DOI: 10.1021/bi501198f

Date:

07-Jan-15 Release date: 21-Jan-15

Protein chain

P19821  (DPO1_THEAQ) -  DNA polymerase I, thermostable from Thermus aquaticus

Seq: 832 a.a.

Struc: 539 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

G-A-C-C-A-C-G-G-C-G-C-DOC 12 bases

T-G-G-G-C-G-C-C-G-T-G-G-T-C 14 bases

Enzyme reactions

• Enzyme class: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1021/bi501198f

Biochemistry 54:881-889 (2015)

PubMed id: 25537790

A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.

E.Y.Wu, A.R.Walsh, E.C.Materne, E.P.Hiltner, B.Zielinski, B.R.Miller, L.Mawby, E.Modeste, C.A.Parish, W.M.Barnes, M.B.Kermekchiev.

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.