 |
PDBsum entry 1qtm
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Transferase/DNA
|
PDB id
|
|
|
|
1qtm
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Structure-Based design of taq DNA polymerases with improved properties of dideoxynucleotide incorporation.
|
|
|
Authors
|
|
Y.Li,
V.Mitaxov,
G.Waksman.
|
|
|
Ref.
|
|
Proc Natl Acad Sci U S A, 1999,
96,
9491-9496.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
|
|
|
