PDBsum entry 1s9f

Transferase/DNA

PDB id: 1s9f

Contents

Protein chains

341 a.a. *

DNA/RNA

Ligands

DDY ×4

Metals

_CA ×4

_MG ×4

Waters ×1129

* Residue conservation analysis

PDB id:

1s9f

Name:

Transferase/DNA

Title:

Dpo with at matched

Structure:

5'-d( Gp Gp Gp Gp Gp Ap Ap Gp Gp Ap Cp Tp A)-3'. Chain: e, f, g, h. Engineered: yes. 5'-d( T Tp Cp Ap Gp Tp Ap Gp Tp Cp Cp Tp Tp Cp Cp Cp Cp C)- 3'. Chain: i, j, k, l. Engineered: yes. DNA polymerase iv. Chain: a, b, c, d.

Source:

Synthetic: yes. Sulfolobus solfataricus. Organism_taxid: 2287. Gene: dbh, dpo4, sso2448. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Trimer (from PQS)

Resolution:

2.00Å R-factor: 0.205 R-free: 0.239

Authors:

J.Trincao,R.E.Johnson,W.T.Wolfle,C.R.Escalante,S.Prakash,L.Prakash, A.K.Aggarwal

Key ref:

J.Trincao et al. (2004). Dpo4 is hindered in extending a G.T mismatch by a reverse wobble. Nat Struct Mol Biol, 11, 457-462. PubMed id: 15077104 DOI: 10.1038/nsmb755

Date:

04-Feb-04 Release date: 15-Feb-05

Protein chains

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

T-C-A-G-T-A-G-T-C-C-T-T-C-C-C-C-C 17 bases

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

T-C-A-G-T-A-G-T-C-C-T-T-C-C-C-C-C 17 bases

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

T-C-A-G-T-A-G-T-C-C-T-T-C-C-C-C-C 17 bases

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

T-C-A-G-T-A-G-T-C-C-T-T-C-C-C-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/nsmb755

Nat Struct Mol Biol 11:457-462 (2004)

PubMed id: 15077104

Dpo4 is hindered in extending a G.T mismatch by a reverse wobble.

J.Trincao, R.E.Johnson, W.T.Wolfle, C.R.Escalante, S.Prakash, L.Prakash, A.K.Aggarwal.

ABSTRACT

The ability or inability of a DNA polymerase to extend a mispair directly affects the establishment of genomic mutations. We report here kinetic analyses of the ability of Dpo4, a Y-family polymerase from Sulfolobus solfataricus, to extend from all mispairs opposite a template G or T. Dpo4 is equally inefficient at extending these mispairs, which include, surprisingly, a G.T mispair expected to conform closely to Watson-Crick geometry. To elucidate the basis of this, we solved the structure of Dpo4 bound to G.T-mispaired primer template in the presence of an incoming nucleotide. As a control, we also determined the structure of Dpo4 bound to a matched A-T base pair at the primer terminus. The structures offer a basis for the low efficiency of Dpo4 in extending a G.T mispair: a reverse wobble that deflects the primer 3'-OH away from the incoming nucleotide.

Selected figure(s)

Figure 1.
Figure 1. Dpo4-catalyzed extension of a G T primer -terminal mispair. (a) The incorporation of dGTP opposite C following an A-T primer -terminal base pair (left) is 500-fold more efficient when compared to the incorporation of dGTP opposite C following a G T primer -terminal mispair (right). The incorporation of dGTP (200 M) was examined for the indicated time periods for each substrate. The enzyme concentrations for the A-T base-paired and the G T mispaired primer termini were 0.2 nM and 1.2 nM, respectively. (b) The rate of dGTP incorporation opposite a template C following an A-T primer -terminal base pair (left) or following a G T primer -terminal mispair (right) was graphed as a function of dGTP concentration. The solid line represents the best fit to the Michaelis-Menten equation. The steady-state parameters, k [cat] and K [m], are listed in Table 1.

Figure 2.
Figure 2. Dpo4 in complex with matched and mismatched primer termini. (a) An overall view of different Dpo4 -DNA complexes: the type I structure determined by Yang and colleagues, and the structures reported here of Dpo4 bound to a mismatched (G T) and the matched (A-T) primer template. Dpo4 in each complex is colored by palm (blue), fingers (yellow) and thumb (orange) domains, as well as PAD (green). The terminal base pair of the DNA in each complex is red. (b) Close-up view of the template-primer terminus in each complex, highlighting the terminal base pair (red), the incoming nucleotide, the active site residues (Asp7, Asp105 and Glu106) coordinating a metal ion (yellow ball, refined as Ca^2+), residues (Tyr48, Arg51 and Lys159) bonding to the di- or triphosphate moiety of the incoming nucleotide, as well as residues (Val32, Ala42 and Gly58) from the fingers domain that impinge on the templating base. Also in red is the C3' atom at the primer terminus. The figure was generated with MolMol32 and PovRay (http://www.povray.org). (c) 2F [o] - F [c] electron density for a portion of the primer and the incoming nucleotide in the G T complex. The map was computed with the terminal guanine (ddG) and the incoming nucleotide (ddCTP) omitted (and followed by simulated annealing). The terminal guanine inverts to form a reverse wobble. Also in red is the hypothetical position for the guanine in a standard wobble configuration (which is completely out of density).

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2004, 11, 457-462) copyright 2004.

Figures were selected by an automated process.