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Lookup NU author(s): Gillian Shuttleworth, Professor Bernard Connolly
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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. © 2004 Elsevier Ltd. All rights reserved.
Author(s): Shuttleworth G, Fogg MJ, Kurpiewski MR, Jen-Jacobson L, Connolly BA
Publication type: Article
Publication status: Published
Journal: Journal of Molecular Biology
Year: 2004
Volume: 337
Issue: 3
Pages: 621-634
ISSN (print): 0022-2836
ISSN (electronic): 1089-8638
Publisher: Academic Press
URL: http://dx.doi.org/10.1016/j.jmb.2004.01.021
DOI: 10.1016/j.jmb.2004.01.021
PubMed id: 15019782
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