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 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.
Shuttleworth
Gillian
G
School of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Fogg
Mark J
MJ
Kurpiewski
Michael R
MR
Jen-Jacobson
Linda
L
Connolly
Bernard A
BA
D001105:Archaea / Q000235:genetics*; D001483:Base Sequence; D004261:DNA Replication; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D013698:Templates, Genetic; D013816:Thermodynamics; D014498:Uracil / Q000378:metabolism*
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D014498:Uracil; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase