PDBsum entry 2vwj

DNA replication

PDB id: 2vwj

Contents

Protein chain

757 a.a. *

DNA/RNA

Metals

__K ×3

Waters ×1

* Residue conservation analysis

PDB id:

2vwj

Name:

DNA replication

Title:

Uracil recognition in archaeal DNA polymerases captured by x-ray crystallography.

Structure:

DNA polymerase. Chain: a. Synonym: tgo polymerase DNA complex, to pol. Engineered: yes. Mutation: yes. 5'-d( Ap Ap Up Gp Gp Ap Gp Ap Cp Gp Gp Cp Tp Tp Tp Tp Gp Cp Cp Gp Tp Gp Tp C)-3'. Chain: b. Engineered: yes

Source:

Thermococcus gorgonarius. Organism_taxid: 71997. Expressed in: escherichia coli. Expression_system_taxid: 469008. Synthetic: yes

Resolution:

2.78Å R-factor: 0.229 R-free: 0.264

Authors:

S.J.Firbank,J.Wardle,P.Heslop,R.J.Lewis,B.A.Connolly

Key ref:

S.J.Firbank et al. (2008). Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography. J Mol Biol, 381, 529-539. PubMed id: 18614176 DOI: 10.1016/j.jmb.2008.06.004

Date:

25-Jun-08 Release date: 22-Jul-08

Protein chain

P56689  (DPOL_THEGO) -  DNA polymerase from Thermococcus gorgonarius

Seq: 773 a.a.

Struc: 757 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DNA/RNA chain

A-A-U-G-G-A-G-A-C-A-C-G-G-C-T-T-T-T-G-C-C-G-T-G-T-C 26 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: 10.1016/j.jmb.2008.06.004

J Mol Biol 381:529-539 (2008)

PubMed id: 18614176

Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography.

S.J.Firbank, J.Wardle, P.Heslop, R.J.Lewis, B.A.Connolly.

ABSTRACT

Archaeal family B DNA polymerases bind tightly to template-strand uracil and stall replication on encountering the pro-mutagenic base. This article describes an X-ray crystal structure, at 2.8 A resolution, of Thermococcus gorgonarius polymerase in complex with a DNA primer-template containing uracil in the single-stranded region. The DNA backbone is distorted to position the uracil deeply within a pocket, located in the amino-terminal domain of the polymerase. Specificity arises from a combination of hydrogen bonds between the protein backbone and uracil, with the pocket shaped to prevent the stable binding of the four standard DNA bases. Strong interactions are seen with the two phosphates that flank the uracil and the structure gives clues concerning the coupling of uracil binding to the halting of replication. The importance of key amino acids, identified by the analysis of the structure and their conservation between archaeal polymerases, was confirmed by site-directed mutagenesis. The crystal structure of V93Q, a polymerase variant that no longer recognises uracil, is also reported, explaining the V93Q phenotype by the steric exclusion of uracil from the pocket.

Selected figure(s)

Figure 1.
Fig. 1. Structure of Tgo-Pol:DNA complex. (a) The sequence of the primer–template mimic used in this study. Sixteen bases are palindromic and form an 8-bp duplex with four consecutive thymines forming the hairpin. The uracil is + 4 from the duplex–single-stranded junction. (b) Overall fold of Tgo-Pol in complex with uracil-containing DNA. The N-terminal domain (1–130 and 327–368) is coloured yellow, the exonuclease domain (131–326) lilac, the palm (369–449 and 500–585) magenta, the fingers (450–499) cyan, and the thumb (586–773) green. The DNA is red. (c) Overlay of Tgo-Pol:DNA with apo Tgo-Pol. For Tgo-Pol:DNA the protein is yellow with red DNA, apo Tgo-Pol (PDB 1TGO) is teal. Although the majority of the proteins superimpose well (left), the thumb exhibits a rotation upon DNA binding (right). The two images are approximately 90° apart by rotation around the vertical axis. (d) The movement of the thumb domain, relative to the rest of the polymerase, as a consequence of duplex DNA binding. The view is approximately 90° away from that shown in (b) in a clockwise direction around a horizontal axis. Apo Tgo-Pol (PDB 1TGO) is the left image, the structure with DNA the right. The interaction with DNA causes repositioning of the thumb, greatly reducing the contact area between the thumb and exonuclease domains. The domains are coloured as in (b).

Figure 2.
Fig. 2. DNA and the contacts it makes to Tgo-Pol. (a) Final 2F[obs] − F[calc] electron density map (gold) contoured at 1σ for the primer–template region of the DNA (red) illustrating the quality of the maps for the majority of the DNA component of the structure. (b) Contacts observed between Tgo-Pol and DNA. The same colour scheme as in Fig. 1 is used to identify individual domains. Most of the contacts (dotted lines) are polar in nature, the exception being the hydrophobic stacking between V93 and the uracil base (continuous line). The specific interactions with uracil are shown in more detail in Fig. 4.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 381, 529-539) copyright 2008.

Figures were selected by an automated process.