1X9W

T7 DNA polymerase in complex with a primer/template DNA containing a disordered N-2 aminofluorene on the template, crystallized with dideoxy-ATP as the incoming nucleotide.

Summary for 1X9W

• Entry DOI: 10.2210/pdb1x9w/pdb
• Related: 1T7P 1X9M 1X9S
• Descriptor: 5'-D(*GP*GP*AP*GP*AP*GP*TP*GP*AP*TP*T*GP*GP*T*AP*GP*TP*GP*TP*GP*AP*(2DT))-3', 5'-D(*CP*CP*CP*(AFG)*AP*TP*CP*AP*CP*AP*CP*TP*AP*CP*CP*AP*AP*TP*CP*AP*CP*TP*CP*TP*CP*C)-3', DNA polymerase, ... (6 entities in total)
• Functional Keywords: dna polymerase, n-2-aminofluorene, mutagenesis, replication block, transferase-electron transport-dna complex, transferase/electron transport/dna
• Biological source: Enterobacteria phage T7; More
• Total number of polymer chains: 4
• Total formula weight: 105664.21

Authors

Dutta, S.,Li, Y.,Johnson, D.,Dzantiev, L.,Richardson, C.C.,Romano, L.J.,Ellenberger, T. (deposition date: 2004-08-24, release date: 2004-10-26, Last modification date: 2024-02-14)

Primary citation

Dutta, S.,Li, Y.,Johnson, D.,Dzantiev, L.,Richardson, C.C.,Romano, L.J.,Ellenberger, T.
Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis.
Proc.Natl.Acad.Sci.Usa, 101:16186-16191, 2004

PubMed 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.

PubMed: 15528277
DOI: 10.1073/pnas.0406516101

Experimental method

X-RAY DIFFRACTION (2.3 Å)