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Interplay between DNA Polymerase and Proliferating Cell Nuclear Antigen Switches Off Base Excision Repair of Uracil and Hypoxanthine during Replication in Archaea

Lookup NU author(s): Kieran Emptage, Dr Rory O'Neill, Dr Alexandra Solovyova, Professor Bernard Connolly

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Abstract

Archaeal family-B DNA polymerases bind tightly to uracil and hypoxanthine (the deamination products of cytosine and adenine), resulting in profound inhibition of DNA replication. Investigation of the mechanism of inhibition, using single-turnover kinetics with polymerase in excess of DNA, indicated that deoxy-NTPs were efficiently bound to the polymerase-DNA complex but very poorly incorporated into the extending chain. Addition of the processivity factor proliferating cell nuclear antigen (PCNA) resulted in increased affinity of the polymerase for all primer-templates, producing extremely tight complexes when uracil (Kd = 16 pM) or hypoxanthine (Kd = 65 pM) was present. Analytical ultracentrifugation confirmed the stability of these complexes and revealed a polymerase/PCNA/DNA stoichiometry of 1:1:1. However, PCNA had no influence on the ability of the polymerase to read through uracil and hypoxanthine, the same kinetic parameters being observed with or without the processivity factor. The specificity constants determined using single-turnover kinetics showed that uracil and hypoxanthine slowed the polymerase by factors of ∼ 5000 and 3000, respectively. Uracil and hypoxanthine are removed from DNA by base excision repair, initiated by uracil-DNA glycosylase and endonuclease V, respectively. Both enzymes are profoundly inhibited by the simultaneous binding of both PCNA and polymerase to primer-templates, with polymerase alone being much less effective. Thus, when the PCNA-polymerase complex encounters uracil/hypoxanthine in DNA templates, base excision repair is switched off, protecting the complex from a repair pathway that is dangerous in the context of single-stranded DNA formed during replication. © 2008 Elsevier Ltd. All rights reserved.

Publication metadata

Author(s): Emptage K, O'Neill R, Solovyova A, Connolly B

Publication type: Article

Publication status: Published

Journal: Journal of Molecular Biology

Year: 2008

Volume: 383

Issue: 4

Pages: 762-771

ISSN (print): 0022-2836

ISSN (electronic): 1089-8638

Publisher: Academic Press

URL: http://dx.doi.org/10.1016/j.jmb.2008.08.018

DOI: 10.1016/j.jmb.2008.08.018

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