PDBsum entry 1q9y

Transferase, replication/DNA

PDB id: 1q9y

Contents

Protein chain

902 a.a. *

DNA/RNA

Ligands

DCP

Metals

_CA ×3

Waters ×84

* Residue conservation analysis

PDB id:

1q9y

Name:

Transferase, replication/DNA

Title:

Crystal structure of enterobacteria phage rb69 gp43 DNA polymerase complexed with 8-oxoguanosine containing DNA

Structure:

5'-ac(8-oxoguanosine)ggtaagcagtccgcg-3'. Chain: t. Engineered: yes. 5'-gcggactgcttac(dideoxycytidine)-3'. Chain: p. Engineered: yes. DNA polymerase. Chain: a. Synonym: gp43.

Source:

Synthetic: yes. Other_details: DNA synthesizer. Enterobacteria phage rb69. Organism_taxid: 12353. Gene: 43. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

2.80Å R-factor: 0.207 R-free: 0.271

Authors:

E.Freisinger,A.P.Grollman,H.Miller,C.Kisker

Key ref:

E.Freisinger et al. (2004). Lesion (in)tolerance reveals insights into DNA replication fidelity. EMBO J, 23, 1494-1505. PubMed id: 15057282 DOI: 10.1038/sj.emboj.7600158

Date:

26-Aug-03 Release date: 27-Apr-04

Protein chain

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

Seq: 903 a.a.

Struc: 902 a.a.*

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

DNA/RNA chains

A-C-8OG-G-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-C-DOC 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

DOI no: 10.1038/sj.emboj.7600158

EMBO J 23:1494-1505 (2004)

PubMed id: 15057282

Lesion (in)tolerance reveals insights into DNA replication fidelity.

E.Freisinger, A.P.Grollman, H.Miller, C.Kisker.

ABSTRACT

The initial encounter of an unrepaired DNA lesion is likely to be with a replicative DNA polymerase, and the outcome of this event determines whether an error-prone or error-free damage avoidance pathway is taken. To understand the atomic details of this critical encounter, we have determined the crystal structures of the pol alpha family RB69 DNA polymerase with DNA containing the two most prevalent, spontaneously generated premutagenic lesions, an abasic site and 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG). Identification of the interactions between these damaged nucleotides and the active site provides insight into the capacity of the polymerase to incorporate a base opposite the lesion. A novel open, catalytically inactive conformation of the DNA polymerase has been identified in the complex with a primed abasic site template. This structure provides the first molecular characterization of the DNA synthesis barrier caused by an abasic site and suggests a general mechanism for polymerase fidelity. In contrast, the structure of the ternary 8-oxodG:dCTP complex is almost identical to the replicating complex containing unmodified DNA, explaining the relative ease and fidelity by which this lesion is bypassed.

Selected figure(s)

Figure 1.
Figure 1 Oligonucleotide sequences and DNA adducts. (A) The 14 nt primers are identical in all trials and terminated by ddC (indicated by C^*). Template strands are 18 nt long with a 3'-dG overhang. X denotes the position of the lesion for primer/template combinations (1) and (2), and the arrow the position of dNTP incorporation. (B) Structures of the lesions at position X in the templates 8-oxodG and tetrahydrofuran (abasic site model).

Figure 7.
Figure 7 Influence of Gly 568 on DNA binding to the polymerase active site. Column (I) shows the event of nucleotide insertion opposite an unmodified template strand with adenine in the active site, while column (II) depicts the case of a template containing an abasic site as in the AP:dG complex. Vertical arrows specify the strained (red) or the relaxed state (green), respectively. Diagonal arrows indicate whether the polymerase is in the closed (red) or open conformation (green). The template strand is depicted in magenta and the incoming nucleotide in green. The yellow box indicates the position of Gly 568. (IA) and (IIA) show the polymerase in the strained state and the open conformation. Transition into the relaxed state presumably causes the adenine base of the unmodified template to be pushed back (IB), while the AP-containing template is unaffected (IIB). (IC) and (IIC) depict an incoming dNTP bound to the base of the fingers domains. Transition to the closed and strained conformation ensures the correct positioning of all residues to enable the catalytic phosphodiester bond formation (ID). In the case of AP, a closed and relaxed conformation is feasible (IID). The missing complementary base causes the dNTP to be held in place less tightly and phosphodiester bond formation is less efficient (IIE).

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2004, 23, 1494-1505) copyright 2004.

Figures were selected by an automated process.