PDBsum entry 1qtm

Transferase/DNA

PDB id: 1qtm

Contents

Protein chain

539 a.a. *

DNA/RNA

Ligands

TTP

Metals

_MG ×2

Waters ×165

* Residue conservation analysis

PDB id:

1qtm

Name:

Transferase/DNA

Title:

Ddttp-trapped closed ternary complex of the large fragment of DNA polymerase i from thermus aquaticus

Structure:

5'-d( Gp Ap Cp Cp Ap Cp Gp Gp Cp Gp Cp (2Dt))-3'. Chain: b. Engineered: yes. 5'-d( Ap Ap Ap Gp Cp Gp Cp Cp Gp Tp Gp Gp Tp C)-3'. Chain: c. Engineered: yes. DNA polymerase i. Chain: a. Fragment: klenow fragment, residues 293-831.

Source:

Synthetic: yes. Thermus aquaticus. Organism_taxid: 271. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

2.30Å R-factor: 0.227 R-free: 0.280

Authors:

Y.Li,V.Mitaxov,G.Waksman

Key ref:

Y.Li et al. (1999). Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. Proc Natl Acad Sci U S A, 96, 9491-9496. PubMed id: 10449720 DOI: 10.1073/pnas.96.17.9491

Date:

28-Jun-99 Release date: 16-Aug-99

Protein chain

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

Seq: 832 a.a.

Struc: 539 a.a.

DNA/RNA chains

G-A-C-C-A-C-G-G-C-G-C-2DT 12 bases

A-A-A-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: 10.1073/pnas.96.17.9491

Proc Natl Acad Sci U S A 96:9491-9496 (1999)

PubMed id: 10449720

Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation.

Y.Li, V.Mitaxov, G.Waksman.

ABSTRACT

The Taq DNA polymerase is the most commonly used enzyme in DNA sequencing. However, all versions of Taq polymerase are deficient in two respects: (i) these enzymes incorporate each of the four dideoxynucleoside 5' triphosphates (ddNTPs) at widely different rates during sequencing (ddGTP, for example, is incorporated 10 times faster than the other three ddNTPs), and (ii) these enzymes show uneven band-intensity or peak-height patterns in radio-labeled or dye-labeled DNA sequence profiles, respectively. We have determined the crystal structures of all four ddNTP-trapped closed ternary complexes of the large fragment of the Taq DNA polymerase (Klentaq1). The ddGTP-trapped complex structure differs from the other three ternary complex structures by a large shift in the position of the side chain of residue 660 in the O helix, resulting in additional hydrogen bonds being formed between the guanidinium group of this residue and the base of ddGTP. When Arg-660 is mutated to Asp, Ser, Phe, Tyr, or Leu, the enzyme has a marked and selective reduction in ddGTP incorporation rate. As a result, the G track generated during DNA sequencing by these Taq polymerase variants does not terminate prematurely, and higher molecular-mass G bands are detected. Another property of these Taq polymerase variants is that the sequencing patterns produced by these enzymes are remarkably even in band-intensity and peak-height distribution, thus resulting in a significant improvement in the accuracy of DNA sequencing.

Selected figure(s)

Figure 1.
Fig. 1. Stereo diagram of the interactions between the side chain of Arg-660 and the incoming base. In the protein, only the O helix is represented and is colored in red, gold, dark blue, and dark green for the ddCTP-, ddATP-, ddTTP-, and ddGTP-trapped complexes, respectively. In the DNA, only the dCMP/ddGTP pair is represented and is colored in green. H-bond interactions between Arg-660 and the O6 and N7 atoms in the base of the ddGTP are indicated by lines. Distances between atoms involved in H-bonds are indicated.

Figure 2.
Fig. 2. Comparison of DNA sequencing by Taq-WT, Taq-RD, Taq-RL, Taq-RY, Taq-RS, and Taq-RF. The enzymes used for sequencing are indicated above the corresponding sequences.

Figures were selected by an automated process.