PDBsum entry 3lzi

Transferase/DNA

PDB id: 3lzi

Contents

Protein chain

903 a.a. *

DNA/RNA

Ligands

DTP

Metals

_CA ×5

Waters ×176

* Residue conservation analysis

PDB id:

3lzi

Name:

Transferase/DNA

Title:

Rb69 DNA polymerase (y567a) ternary complex with datp opposite 7,8- dihydro-8-oxoguanine

Structure:

DNA polymerase. Chain: a. Synonym: gp43. Engineered: yes. Mutation: yes. DNA (5'-d(p Tp Cp Ap (8Og) p Gp Tp Ap Ap Gp Cp Ap Gp Tp Cp Cp Gp Cp G)-3'). Chain: t. Engineered: yes.

Source:

Enterobacteria phage rb69. Bacteriophage rb69. Organism_taxid: 12353. Gene: 43, gp43. Expressed in: escherichia coli. Expression_system_taxid: 469008. Synthetic: yes. Synthetic: yes

Resolution:

2.30Å R-factor: 0.208 R-free: 0.258

Authors:

M.Wang,J.Beckman,G.Blaha,J.Wang,W.H.Konigsberg

Key ref:

J.Beckman et al. (2010). Substitution of Ala for Tyr567 in RB69 DNA polymerase allows dAMP to be inserted opposite 7,8-dihydro-8-oxoguanine . Biochemistry, 49, 4116-4125. PubMed id: 20411947

Date:

01-Mar-10 Release date: 12-May-10

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 3 residue positions (black crosses)

DNA/RNA chains

T-C-A-8OG-G-T-A-A-G-C-A-G-T-C-C-G-C-G 18 bases

G-C-G-G-A-C-T-G-C-T-T-A-DOC 13 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 49:4116-4125 (2010)

PubMed id: 20411947

Substitution of Ala for Tyr567 in RB69 DNA polymerase allows dAMP to be inserted opposite 7,8-dihydro-8-oxoguanine .

J.Beckman, M.Wang, G.Blaha, J.Wang, W.H.Konigsberg.

ABSTRACT

Accurate copying of the genome by DNA polymerases is challenging due in part to the continuous damage inflicted on DNA, which results from its contact with reactive oxygen species (ROS), producing lesions such as 7,8-dihydro-8-oxoguanine (8-oxoG). The deleterious effects of 8-oxoG can be attributed to its dual coding potential that leads to G --> T transversions. The wild-type (wt) pol alpha family DNA polymerase from bacteriophage RB69 (RB69pol) prefers to insert dCMP as opposed to dAMP when situated opposite 8-oxoG by >2 orders of magnitude as demonstrated using pre-steady-state kinetics (k(pol)/K(d,app)). In contrast, the Y567A mutant of RB69pol inserts both dCMP and dAMP opposite 8-oxoG rapidly and with equal efficiency. We have determined the structures of preinsertion complexes for the Y567A mutant with dATP and dCTP opposite a templating 8-oxoG in a 13/18mer primer-template (P/T) at resolutions of 2.3 and 2.1 A, respectively. Our structures show that the 8-oxoG residue is in the anti conformation when paired opposite dCTP, but it flips to a syn conformation forming a Hoogstein base pair with an incoming dATP. Although the Y567A substitution does not significantly change the volume of the pocket occupied by anti-8-oxoG, it does provide residue G568 the flexibility to move deeper into the minor groove of the P/T to accommodate, and stabilize, syn-8-oxoG. These results support the hypothesis that it is the flexibility of the nascent base pair binding pocket (NBP) in the Y567A mutant that allows efficient insertion of dAMP opposite 8-oxoG.