PDBsum entry 2imw

Transferase/DNA

PDB id: 2imw

Contents

Protein chain

348 a.a. *

DNA/RNA

Ligands

DDS

EDO ×6

Metals

_CA ×2

Waters ×240

* Residue conservation analysis

PDB id:

2imw

Name:

Transferase/DNA

Title:

Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase

Structure:

5'-d( Gp Gp Gp Gp Gp Ap Ap Gp Gp Ap Tp Tp C)-3'. Chain: s. Engineered: yes. 5'-d( Tp Ap Gp Ap Ap Tp Cp Cp Tp Tp Cp Cp Cp Cp C)-3'. Chain: t. Engineered: yes. DNA polymerase iv. Chain: p. Synonym: pol iv.

Source:

Synthetic: yes. Sulfolobus solfataricus. Organism_taxid: 2287. Gene: dbin. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.05Å R-factor: 0.232 R-free: 0.284

Authors:

H.Ling,W.Yang

Key ref:

K.A.Fiala et al. (2007). Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase. J Mol Biol, 365, 590-602. PubMed id: 17095011 DOI: 10.1016/j.jmb.2006.10.008

Date:

05-Oct-06 Release date: 16-Jan-07

Protein chain

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

Seq: 352 a.a.

Struc: 348 a.a.

DNA/RNA chains

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

T-A-G-A-A-T-C-C-T-T-C-C-C-C-C 15 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: 10.1016/j.jmb.2006.10.008

J Mol Biol 365:590-602 (2007)

PubMed id: 17095011

Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase.

K.A.Fiala, J.A.Brown, H.Ling, A.K.Kshetry, J.Zhang, J.S.Taylor, W.Yang, Z.Suo.

ABSTRACT

Numerous template-dependent DNA polymerases are capable of catalyzing template-independent nucleotide additions onto blunt-end DNA. Such non-canonical activity has been hypothesized to increase the genomic hypermutability of retroviruses including human immunodeficiency viruses. Here, we employed pre-steady state kinetics and X-ray crystallography to establish a mechanism for blunt-end additions catalyzed by Sulfolobus solfataricus Dpo4. Our kinetic studies indicated that the first blunt-end dATP incorporation was 80-fold more efficient than the second, and among natural deoxynucleotides, dATP was the preferred substrate due to its stronger intrahelical base-stacking ability. Such base-stacking contributions are supported by the 41-fold higher ground-state binding affinity of a nucleotide analog, pyrene nucleoside 5'-triphosphate, which lacks hydrogen bonding ability but possesses four conjugated aromatic rings. A 2.05 A resolution structure of Dpo4*(blunt-end DNA)*ddATP revealed that the base and sugar of the incoming ddATP, respectively, stack against the 5'-base of the opposite strand and the 3'-base of the elongating strand. This unprecedented base-stacking pattern can be applied to subsequent blunt-end additions only if all incorporated dAMPs are extrahelical, leading to predominantly single non-templated dATP incorporation.

Selected figure(s)

Figure 4.
Figure 4. Crystal structure of Dpo4•blunt-end X-1•ddATP (2.05 Å). (a) Overall ternary structure. Dpo4 was shown in grey ribbons while DNA and ddATP were shown as ball-and-stick models. The ddATP is highlighted in magenta. The Ca^2+ ion was shown in a green sphere. (b) The zoomed in view of the active site including ddATP and the blunt-end base-pair. The residues in contact with ddATP were shown as ball-and-stick models (grey for atom C, red for atom O, yellow for atom S). Only the side chain and main chain atoms involved were shown. (c) 2F[o] - F[c] electron density map contoured at 1.2 σ (light-blue) of the active site. The amino acid residues, two blunt-end DNA base-pairs, and incoming ddATP were shown as ball-and-stick models. Figure 4. Crystal structure of Dpo4•blunt-end X-1•ddATP (2.05 Å). (a) Overall ternary structure. Dpo4 was shown in grey ribbons while DNA and ddATP were shown as ball-and-stick models. The ddATP is highlighted in magenta. The Ca^2+ ion was shown in a green sphere. (b) The zoomed in view of the active site including ddATP and the blunt-end base-pair. The residues in contact with ddATP were shown as ball-and-stick models (grey for atom C, red for atom O, yellow for atom S). Only the side chain and main chain atoms involved were shown. (c) 2F[o] - F[c] electron density map contoured at 1.2 σ (light-blue) of the active site. The amino acid residues, two blunt-end DNA base-pairs, and incoming ddATP were shown as ball-and-stick models.

Figure 5.
Figure 5. Proposed mechanisms of blunt-end additions of (a) dPTP and (b) dATP. dATP and dPTP are represented by A and P in different colors, respectively. The Watson-Crick hydrogen bonds were drawn as dashed lines while the base-stacking interactions were shadowed in green. The stacking interactions between the 2′-deoxyribose (R) of an incoming nucleotide and the 5′-terminal base A are displayed in a green box. The van der Waals interactions between an incoming nucleotide and Dpo4 active site residues were not shown for clarity. Figure 5. Proposed mechanisms of blunt-end additions of (a) dPTP and (b) dATP. dATP and dPTP are represented by A and P in different colors, respectively. The Watson-Crick hydrogen bonds were drawn as dashed lines while the base-stacking interactions were shadowed in green. The stacking interactions between the 2′-deoxyribose (R) of an incoming nucleotide and the 5′-terminal base A are displayed in a green box. The van der Waals interactions between an incoming nucleotide and Dpo4 active site residues were not shown for clarity.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 365, 590-602) copyright 2007.

Figures were selected by an automated process.