Recognition of the Pro-mutagenic Base Uracil by Family B DNA Polymerases from Archaea

Recognition of the Pro-mutagenic Base Uracil by Family B DNA Polymerases from Archaea

Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique “template-strand proof-reading” has been studied using equilibrium binding measurements, DNA footprinting, van't Hof...

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Bibliographic Details
Published in:Journal of molecular biology Vol. 337; no. 3; pp. 621 - 634
Main Authors: Shuttleworth, Gillian, Fogg, Mark J., Kurpiewski, Michael R., Jen-Jacobson, Linda, Connolly, Bernard A.
Format: Journal Article
Language:English
Published: England Elsevier Ltd 26-03-2004
Subjects:
Archaea
Archaea - genetics
Base Sequence
DNA polymerase
DNA Replication
DNA, Single-Stranded
DNA-Directed DNA Polymerase - metabolism
DNA-repair
Mutagenesis
Oligodeoxyribonucleotides - metabolism
Protein Binding
read-ahead
Templates, Genetic
Thermodynamics
uracil
Uracil - metabolism
uracil
DNA polymerase
read-ahead
DNA-repair
archaea
Pfu-Pol, Pyrococcus furiosus polymerase
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Summary:Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique “template-strand proof-reading” has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer–templates that contain uracil four bases in front of the primer–template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer–templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a “read-ahead” mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.
Bibliography:ObjectType-Article-2
SourceType-Scholarly Journals-1
ObjectType-Feature-1
content type line 23
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2004.01.021