PDBsum entry 2agq

Transferase/DNA

PDB id: 2agq

Contents

Protein chain

341 a.a. *

DNA/RNA

Ligands

DTP

Metals

_CA ×2

_MG

Waters ×160

* Residue conservation analysis

PDB id:

2agq

Name:

Transferase/DNA

Title:

Fidelity of dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis

Structure:

5'-d( Gp Gp Cp Tp Ap Cp Ap Gp Gp Ap Cp Tp (Doc))-3'. Chain: b. Engineered: yes. 5'-d( Tp Cp Ap Tp Gp Ap Gp Tp Cp Cp Tp Gp Tp Ap Gp Cp C)- 3'. Chain: c. Engineered: yes. DNA polymerase iv. Chain: a.

Source:

Synthetic: yes. Other_details: chemically synthesized. Sulfolobus solfataricus. Organism_taxid: 2287. Gene: dbh, dpo4. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Trimer (from PQS)

Resolution:

2.10Å R-factor: 0.227 R-free: 0.254

Authors:

H.Ling,W.Yang

Key ref:

A.Vaisman et al. (2005). Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis. EMBO J, 24, 2957-2967. PubMed id: 16107880 DOI: 10.1038/sj.emboj.7600786

Date:

27-Jul-05 Release date: 06-Sep-05

Protein chain

Q97W02  (DPO4_SULSO) -  DNA polymerase IV from Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)

Seq: 352 a.a.

Struc: 341 a.a.

DNA/RNA chains

G-G-C-T-A-C-A-G-G-A-C-T-DOC 13 bases

T-C-A-T-G-A-G-T-C-C-T-G-T-A-G-C-C 17 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: 10.1038/sj.emboj.7600786

EMBO J 24:2957-2967 (2005)

PubMed id: 16107880

Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis.

A.Vaisman, H.Ling, R.Woodgate, W.Yang.

ABSTRACT

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.

Selected figure(s)

Figure 1.
Figure 1 Ribbon diagrams of the T/dATP (A), T/dGTP-1 (B) and T/dGTP-2 (C) and T/G (D) structures around the active site. Dpo4 is shown as ribbons. The three conserved carboxylates in the active site, the last two base pairs of the primer/template and the replicating base pair are shown as ball-and-stick models. The template strand is shown in blue and the primer strand in purple. The incoming nucleotide is shown in different colors for each crystal structure. The metal ions are shown as green spheres. The 2F[o]-F[c] electron density maps are contoured at 1 level and superimposed onto the nucleic acid portion.

Figure 2.
Figure 2 Structural comparison of Dpo4 and T7 DNA polymerase. (A) The replicating base pairs in three Dpo4 structures (T/dGTP, T/dATP and Ab-2A) are shown as ball-and-stick models. The two metal ions (A and B) are shown as green spheres. The A-metal ion position differs in each structure. The conformation of the triphosphate is denoted as 'chair-like' and 'goat tail-like'. (B) Superposition of T/dATP, T/dGTP and Ab-2A structures. The C traces, DNA and nucleotide substrate are shown in stick models. A zoom-in stereo view of the finger domain with the replicating base pair and metal ions (outlined in gray) is shown on the right. The colors representing each structure are indicated. (C) Superposition of the metal ion coordination in Dpo4 (Ab-2A, yellow and brown colors) and T7 DNA polymerase (PDB: 1T7P, blue and green colors) in a stereo view. The oxygen atoms of the three conserved carboxylates and those involved in metal ion coordination are highlighted in red. The metal ion coordination is schematically drawn on the right. Red indicates ligands conserved in both polymerases, light green in Dpo4 only and blue in T7 only. The hypothesized 3'-OH of the primer strand is shown in gray.

The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2005, 24, 2957-2967) copyright 2005.

Figures were selected by an automated process.