PDBsum entry 2w8k

Transferase/DNA

PDB id: 2w8k

Contents

Protein chain

343 a.a. *

DNA/RNA

Ligands

DGT

Metals

_MG ×3

Waters ×80

* Residue conservation analysis

PDB id:

2w8k

Name:

Transferase/DNA

Title:

Y-family DNA polymerase dpo4 bypassing n2-naphthyl-guanine adduct in syn orientation

Structure:

DNA polymerase iv. Chain: a. Engineered: yes. Other_details: y-family DNA polymerase from sulfolobus solfataricus. 5'-d( Gp Gp Gp Gp Gp Ap Ap Gp Gp Ap Tp Tp Cp Doc)-3'. Chain: p. Engineered: yes. Other_details: 14 base primer DNA 5'-ggg gga agg att cc-3'. 5'-d( Tp Cp Ap Cp N2gp Gp Ap Ap Tp Cp Cp

Source:

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

Resolution:

3.10Å R-factor: 0.233 R-free: 0.282

Authors:

R.L.Eoff,H.Zhang,M.Egli,F.P.Guengerich

Key ref:

H.Zhang et al. (2009). Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translesion synthesis past bulky N2-alkylguanine adducts. J Biol Chem, 284, 3563-3576. PubMed id: 19059910 DOI: 10.1074/jbc.M807778200

Date:

16-Jan-09 Release date: 27-Jan-09

Supersedes:

2v4s

Protein chain

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

Seq: 352 a.a.

Struc: 343 a.a.

DNA/RNA chains

G-G-G-G-G-A-A-G-G-A-T-T-C-DOC 14 bases

A-C-N2G-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.M807778200

J Biol Chem 284:3563-3576 (2009)

PubMed id: 19059910

Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translesion synthesis past bulky N2-alkylguanine adducts.

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

ABSTRACT

In contrast to replicative DNA polymerases, Sulfolobus solfataricus Dpo4 showed a limited decrease in catalytic efficiency (k(cat)/Km) for insertion of dCTP opposite a series of N2-alkylguanine templates of increasing size from (methyl (Me) to (9-anthracenyl)-Me (Anth)). Fidelity was maintained with increasing size up to (2-naphthyl)-Me (Naph). The catalytic efficiency increased slightly going from the N2-NaphG to the N2-AnthG substrate, at the cost of fidelity. Pre-steady-state kinetic bursts were observed for dCTP incorporation throughout the series (N2-MeG to N2-AnthG), with a decrease in the burst amplitude and k(pol), the rate of single-turnover incorporation. The pre-steady-state kinetic courses with G and all of the six N2-alkyl G adducts could be fit to a general DNA polymerase scheme to which was added an inactive complex in equilibrium with the active ternary Dpo4.DNA.dNTP complex, and only the rates of equilibrium with the inactive complex and phosphodiester bond formation were altered. Two crystal structures of Dpo4 with a template N2-NaphG (in a post-insertion register opposite a 3'-terminal C in the primer) were solved. One showed N2-NaphG in a syn conformation, with the naphthyl group located between the template and the Dpo4 "little finger" domain. The Hoogsteen face was within hydrogen bonding distance of the N4 atoms of the cytosine opposite N2-NaphG and the cytosine at the -2 position. The second structure showed N2-Naph G in an anti conformation with the primer terminus largely disordered. Collectively these results explain the versatility of Dpo4 in bypassing bulky G lesions.

Selected figure(s)

Figure 7.
Comparison of the Npg-1 and Npg-2 structures. A, superimposition of the refined Npg-1 (cyan) and Npg-2 (purple) structures reveals similar overall topology (r.m.s.d. 0.52). B, the DNA substrates from Npg-1 and Npg-2 were superimposed with the DNA from the ternary complex of Dpo4 inserting dCTP opposite 8-oxoG (orange, pdb ID code 2c2e). A more pronounced bending of the helical axis was observed in the N^2-NaphG-modified DNA. Other perturbations in the N^2-NaphG DNA, e.g. buckling of the bases and widening of the major groove near the primer·template junction, are also evident. C, the active site of the Npg-1 crystal structure is shown in schematic form. The 3F[o] – 2F[c] electron density map (cyan mesh) is contoured at the 1σ level around the N^2-NaphG residue. The incoming dGTP (red) is shown along with the magnesium ions (green spheres). D, base pairing and stacking interactions observed in the Npg-1 structure. E, the active site of the Npg-1 crystal structure is shown in schematic form. The 3F[o] – 2F[c] electron density map (purple mesh) is contoured at the 1σ level around the N^2-NaphG residue. The incoming dGTP (red) is shown along with the magnesium ions (green spheres). F, base pairing and stacking interactions observed in the Npg-2 structure.

Figure 9.
Structural comparison between Dpo4-catalyzed bypass of N^2-NaphG and human pol κ. A, superimposition of the Npg-1 complex (cyan) and the human pol κ ternary complex (purple; pdb ID code 2oh2; r.m.s.d. = 5.35). B, the active site DNA residues from the Npg-1 complex (cyan) are shown along with the superimposed pol κ structure (purple, space-filling). Hydrophobic and/or aromatic residues near the naphthyl moiety are highlighted for both enzymes.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 3563-3576) copyright 2009.

Figures were selected by an automated process.