PDBsum entry 1tk0

Transferase/electron transport/DNA

PDB id: 1tk0

Contents

Protein chains

686 a.a. *

105 a.a. *

DNA/RNA

Ligands

SO4 ×2

DCT

1PE

MES

Metals

_MG ×3

Waters ×533

* Residue conservation analysis

PDB id:

1tk0

Name:

Transferase/electron transport/DNA

Title:

T7 DNA polymerase ternary complex with 8 oxo guanosine and ddctp at the insertion site

Structure:

5'-d( Cp Gp Ap Ap Ap Ap Cp Gp A Cp Gp Gp Cp Cp Ap Gp Tp Gp Cp Cp Ap (Ddg))-3'. Chain: p. Engineered: yes. Other_details: primer strand. 5'-d( Cp Cp Cp (8Og) p Cp Tp Gp Gp Cp Ap Cp Tp Gp Gp Cp Cp Gp Tp Cp Gp Tp Tp Tp Tp Cp G)- 3'. Chain: t.

Source:

Synthetic: yes. Enterobacteria phage t7. Organism_taxid: 10760. Gene: 5. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Escherichia coli. Organism_taxid: 562. Gene: trxa, tsnc, fipa, b3781, c4701, z5291, ecs4714, stm3915,

Biol. unit:

Tetramer (from PQS)

Resolution:

2.30Å R-factor: 0.217 R-free: 0.263

Authors:

L.G.Brieba,B.F.Eichman,R.J.Kokoska,S.Doublie,T.A.Kunkel,T.Ellenberger

Key ref:

L.G.Brieba et al. (2004). Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase. EMBO J, 23, 3452-3461. PubMed id: 15297882 DOI: 10.1038/sj.emboj.7600354

Date:

07-Jun-04 Release date: 31-Aug-04

Protein chain

P00581  (DPOL_BPT7) -  DNA-directed DNA polymerase from Escherichia phage T7

Seq: 704 a.a.

Struc: 686 a.a.

Protein chain

P0AA25  (THIO_ECOLI) -  Thioredoxin 1 from Escherichia coli (strain K12)

Seq: 109 a.a.

Struc: 105 a.a.

DNA/RNA chains

C-G-G-C-C-A-G-T-G-C-C-A-DDG 13 bases

C-8OG-C-T-G-G-C-A-C-T-G-G-C-C-G-T 16 bases

Enzyme reactions

• Enzyme class 2: Chain A: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
• Enzyme class 3: Chain A: E.C.3.1.11.- - ?????
• Enzyme class 4: Chain B: E.C.?

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

Added reference

DOI no: 10.1038/sj.emboj.7600354

EMBO J 23:3452-3461 (2004)

PubMed id: 15297882

Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase.

L.G.Brieba, B.F.Eichman, R.J.Kokoska, S.Doublié, T.A.Kunkel, T.Ellenberger.

ABSTRACT

Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase bypasses the common oxidative lesion 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn).dA (anti) in a Hoogsteen-like base pair at the 3' terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG.dA mismatch, thus providing insight into the high mutagenic potential of 8oG.

Selected figure(s)

Figure 2.
Figure 2 T7 DNA polymerase bypass of an 8oG lesion. Primer extension reactions were performed with exo- (left) and wild-type (right) T7 DNA polymerase with undamaged guanine (G) and 8-oxoguanine (8oG) in comparison to controls containing no enzyme. The images shown are for 3 min incubations of reaction mixtures containing 200- to 400-fold excess of DNA over polymerase. The most intense band in each lane is unreacted primer, at least 80% of which remains unextended for all efficiency reactions performed in this study. The location of 8oG within the template strand is as indicated and enhanced images of products using 8oG are shown to the right of the boxed images. The probability of insertion at each template site, listed in percent to the right of each lane, is an average of 7 -16 determinations and is calculated as described previously (Kokoska et al, 2003).

Figure 4.
Figure 4 Comparison of an open 8oG complex and a closed T ddATP insertion complex. The open 8oG complex (red) and a dT dATP insertion complex (gray) were superimposed using C[ ]atoms. The proteins are depicted as cylinders and the DNA as sticks. Both structures are largely similar but they specifically differ in the orientation adopted by their fingers subdomains. In the closed structure, -helices O and O1 pack against the incoming ddATP (blue) and the template thymine, respectively. In the open structure, the fingers move outwards from the palm subdomain, as shown by the 45° rotation of the O and O1 helices relative to the closed conformation. Residue Tyr530, which moves to the position that would correspond to the templating base of the closed complex, has been omitted for clarity. The templating 8oG, the 5' template strand, and residues 532 -536 located at the junction between helices O and O1 are disordered in the open complex. No interpretable electron density is observed for the metal ions or incoming nucleotide.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2004, 23, 3452-3461) copyright 2004.

Figures were selected by an automated process.