PDBsum entry 1x9m

Transferase/electron transport/DNA

PDB id: 1x9m

Contents

Protein chains

675 a.a. *

105 a.a. *

DNA/RNA

Metals

_MG

Waters ×315

* Residue conservation analysis

PDB id:

1x9m

Name:

Transferase/electron transport/DNA

Title:

T7 DNA polymerase in complex with an n-2-acetylaminofluorene-adducted DNA

Structure:

5'-d( Gp Gp Ap Gp Ap Gp Tp Gp Ap Tp Tp Gp Gp Tp Ap Gp Tp Gp Tp Gp Ap (2Dt))-3'. Chain: c. Engineered: yes. Other_details: DNA primer. 5'-d( Cp Cp Cp (8Fg) p Ap Tp Cp Ap Cp Ap Cp Tp Ap Cp Cp Ap Ap Tp Cp Ap Cp Tp Cp Tp Cp C)- 3'. Chain: d.

Source:

Synthetic: yes. Enterobacteria phage t7. Organism_taxid: 10760. Gene: 5. Expressed in: escherichia coli. Expression_system_taxid: 562. Escherichia coli. Organism_taxid: 562. Gene: trxa, tsnc, fipa.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.10Å R-factor: 0.213 R-free: 0.236

Authors:

S.Dutta,Y.Li,D.Johnson,L.Dzantiev,C.C.Richardson,L.J.Romano, T.Ellenberger

Key ref:

S.Dutta et al. (2004). Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis. Proc Natl Acad Sci U S A, 101, 16186-16191. PubMed id: 15528277 DOI: 10.1073/pnas.0406516101

Date:

23-Aug-04 Release date: 26-Oct-04

Protein chain

P00581  (DPOL_BPT7) -  DNA-directed DNA polymerase from Escherichia phage T7

Seq: 704 a.a.

Struc: 675 a.a.

Protein chain

P0AA25  (THIO_ECOLI) -  Thioredoxin 1 from Escherichia coli (strain K12)

Seq: 109 a.a.

Struc: 105 a.a.

DNA/RNA chains

G-T-A-G-T-G-T-G-A-2DT 10 bases

8FG-A-T-C-A-C-A-C-T-A-C-C 12 bases

Enzyme reactions

• Enzyme class 2: Chain A: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
• Enzyme class 3: Chain A: E.C.3.1.11.- - ?????
• Enzyme class 4: Chain B: E.C.?

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1073/pnas.0406516101

Proc Natl Acad Sci U S A 101:16186-16191 (2004)

PubMed id: 15528277

Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis.

S.Dutta, Y.Li, D.Johnson, L.Dzantiev, C.C.Richardson, L.J.Romano, T.Ellenberger.

ABSTRACT

The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G --> T transversions. We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations.

Selected figure(s)

Figure 1.
Fig. 1. Schematic representation of dG-AAF and dG-AF. The anti conformation (a) is energetically favored for unmodified dG. dG-AF (b) can be in anti conformation or syn conformation. However, for dG-AAF (c), the anti conformation is strongly unfavorable because of the steric hindrance between the acetyl group (circled in green) and the sugar moiety (circled in pink).

Figure 2.
Fig. 2. dG-AAF adopts the syn conformation with the fluorene ring intercalated into the polymerase fingers domain. (a) dG-AAF lies outside the polymerase active site, and the fingers domain of the polymerase is in an open conformation. The T7 gene 5 protein (gray) and the thioredoxin (green) processivity factor are shown as ribbons, with the O-helix within the fingers highlighted in red. The primer strand of the DNA is in light red, the template is in yellow, and dG-AAF is in cyan and green. The region around the dG-AAF binding site is circled. (b) Enlarged view of the circled region in a. The fluorene ring (green) of dG-AAF (cyan and green) is inserted into the fingers domain between helices L, O, O1, O2, and P. (c) The simulated annealing omit electron density around the region of the syn dG-AAF of the dG-AAF-containing complex is shown in stereo, contoured at 2.5 above the mean value. (d) Interaction between dG-AAF and the protein. Hydrophobic side chains (gold) of the fingers form a pocket around the fluorene ring. Two charged residues (pink), Arg-566 and Asp-534, form hydrogen bonding interactions with the G base.

Figures were selected by an automated process.