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Nucleic Acids Research Advance Access published online on September 26, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp800
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© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

The 3'–5' proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases

Henry J. Russell, Tomas T. Richardson, Kieran Emptage and Bernard A. Connolly*

Institute of Cell and Molecular Biosciences (ICaMB), University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK

*To whom correspondence should be addressed. Tel: +44 191 222 7371; Fax: +44 191 222 7424; Email: b.a.connolly{at}ncl.ac.uk

Received March 26, 2009. Revised August 25, 2009. Accepted September 11, 2009.

Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3'–5' proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3' base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3'–5' proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases.


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