PDBsum entry 2ozm

Transferase/DNA

PDB id: 2ozm

Contents

Protein chain

903 a.a. *

DNA/RNA

Ligands

N5P

Metals

_MG

Waters ×400

* Residue conservation analysis

PDB id:

2ozm

Name:

Transferase/DNA

Title:

Crystal structure of rb69 gp43 in complex with DNA with 5-nitp opposite an abasic site analog

Structure:

Template DNA. Chain: t. Synonym: gp43. Engineered: yes. Primer DNA. Chain: p. Engineered: yes. DNA polymerase. Chain: a.

Source:

Synthetic: yes. Enterobacteria phage rb69. Organism_taxid: 12353. Gene: 43. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.86Å R-factor: 0.210 R-free: 0.268

Authors:

K.E.Zahn,H.Belrhali,S.S.Wallace,S.Doublie

Key ref:

K.E.Zahn et al. (2007). Caught bending the A-rule: crystal structures of translesion DNA synthesis with a non-natural nucleotide. Biochemistry, 46, 10551-10561. PubMed id: 17718515

Date:

26-Feb-07 Release date: 23-Oct-07

Protein chain

Q38087  (DPOL_BPR69) -  DNA-directed DNA polymerase from Escherichia phage RB69

Seq: 903 a.a.

Struc: 903 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DNA/RNA chains

C-G-3DR-C-T-T-A-T-G-A-C-A-G-C-C-G-C-G 18 bases

G-C-G-G-C-T-G-T-C-A-T-A-A-DDG 14 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

Biochemistry 46:10551-10561 (2007)

PubMed id: 17718515

Caught bending the A-rule: crystal structures of translesion DNA synthesis with a non-natural nucleotide.

K.E.Zahn, H.Belrhali, S.S.Wallace, S.Doublié.

ABSTRACT

Damage to DNA involving excision of the nucleobase at the N-glycosidic bond forms abasic sites. If a nucleotide becomes incorporated opposite an unrepaired abasic site during DNA synthesis, most B family polymerases obey the A-rule and preferentially incorporate dAMP without instruction from the template. In addition to being potentially mutagenic, abasic sites provide strong blocks to DNA synthesis. A previous crystal structure of an exonuclease deficient variant of the replicative B family DNA polymerase from bacteriophage RB69 (RB69 gp43 exo-) illustrated these properties, showing that the polymerase failed to translocate the DNA following insertion of dAMP opposite an abasic site. We examine four new structures depicting several steps of translesion DNA synthesis by RB69 gp43 exo-, employing a non-natural purine triphosphate analogue, 5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is incorporated more efficiently than dAMP opposite abasic sites. Our structures indicate that a dipole-induced dipole stacking interaction between the 5-nitro group and base 3' to the templating lesion explains the enhanced kinetics of 5-NITP. As with dAMP, the DNA fails to translocate following insertion of 5-NIMP, although distortions at the nascent primer terminus contribute less than previously thought in inducing the stall, given that 5-NIMP preserves relatively undistorted geometry at the insertion site following phosphoryl transfer. An open ternary configuration, novel in B family polymerases, reveals an initial template independent binding of 5-NITP adjacent to the active site of the open polymerase, suggesting that closure of the fingers domain shuttles the nucleotide to the active site while testing the substrate against the template.