PDBsum entry 2w9c

Transferase/DNA

PDB id: 2w9c

Contents

Protein chains

342 a.a. *

DNA/RNA

Ligands

TTP ×2

Metals

_MG ×6

Waters ×109

* Residue conservation analysis

PDB id:

2w9c

Name:

Transferase/DNA

Title:

Ternary complex of dpo4 bound to n2,n2-dimethyl-deoxyguanosine modified DNA with incoming dttp

Structure:

DNA polymerase iv. Chain: a. Synonym: pol iv, dpo4. Engineered: yes. Other_details: y-family DNA polymerase from sulfolobus solfataricus. DNA polymerase iv. Chain: b. Synonym: pol iv, dpo4. Engineered: yes.

Source:

Sulfolobus solfataricus. Organism_taxid: 273057. Strain: p2. Atcc: 35092. Expressed in: escherichia coli. Expression_system_taxid: 469008. Synthetic: yes. Synthetic: yes

Resolution:

2.90Å R-factor: 0.252 R-free: 0.288

Authors:

R.L.Eoff,H.Zhang,I.D.Kosekov,C.J.Rizzo,M.Egli,F.P.Guengerich

Key ref:

H.Zhang et al. (2009). Structure-function relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes. J Biol Chem, 284, 17687-17699. PubMed id: 19542237 DOI: 10.1074/jbc.M109014274

Date:

22-Jan-09 Release date: 12-May-09

Protein chains

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

Seq: 352 a.a.

Struc: 342 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

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

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

A-C-O2G-G-A-A-T-C-C-T-T-C-C-C-C-C 16 bases

A-C-O2G-G-A-A-T-C-C-T-T-C-C-C-C-C 16 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: 10.1074/jbc.M109014274

J Biol Chem 284:17687-17699 (2009)

PubMed id: 19542237

Structure-function relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes.

H.Zhang, R.L.Eoff, I.D.Kozekov, C.J.Rizzo, M.Egli, F.P.Guengerich.

ABSTRACT

Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N2,N2-Me2G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N2,N2-Me2G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3'-terminal dideoxycytosine (Cdd) opposite template N2,N2-Me2G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N2,N2-Me2G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N2,N2-Me2G, with a second water-mediated hydrogen bond between the N-3 atom of Cdd and the O-6 atom of N2,N2-Me2G. A crystal structure of Dpo4 with dTTP opposite template N2,N2-Me2G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N2,N2-Me2G compared with mono-substituted N2-alkyl G adducts.

Selected figure(s)

Figure 3.
Crystal structures of Dpo4 bound to N^2,N^2-Me[2]G-modified DNA.A, superimpositions of DMG-1 (red), DMG-2 (molecule A, cyan), and DMG-3 (molecule A, green) reveal overall similarity in Dpo4 structure. B, representative electron density near the active site of Dpo4 in the DMG-1 structure. The 3F[o][]− 2F[c] map (gray mesh) is shown contoured to the 1σ level. The F[o][]− F[c] difference maps are shown contoured to 3σ and −3σ for positive (red mesh) and negative (green mesh) density, respectively. The terminal C[dd] residue is flipped out of base-stacking orientation. but the incoming dGTP forms a Watson-Crick pair with the cytosine to the 5′-side of N^2,N^2-Me[2]G (DMG).

Figure 4.
Structural examination of wobble pairing with N^2,N^2-Me[2]G-modified DNA in Dpo4.A, overall structure of DNA and corresponding electron density observed in the active site of DMG-2 are shown. The orientation of bases observed in molecule A (B) and molecule B (C) of the DMG-2 structure is shown. D, overall structure of DNA and corresponding electron density observed in the active site of DMG-2 are shown. The orientation of bases observed in molecule A (E) and molecule B (F) of the DMG-2 structure is shown. In all panels, the 3F[o][]− 2F[c] map (gray mesh) for DNA bound in the DMG-2 structure is shown contoured to 1σ level (gray mesh) with the F[o][]− F[c] difference maps shown contoured to 3σ and −3σ for positive (red mesh) and negative (green mesh) density, respectively.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 17687-17699) copyright 2009.

Figures were selected by an automated process.