Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil |
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| Authors: | Tomas T Richardson Xiaohua Wu Brian J Keith Pauline Heslop Anita C Jones Bernard A Connolly |
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| Institution: | 1.Institute of Cell and Molecular Biosciences (ICaMB), The University of Newcastle, Newcastle upon Tyne NE2 4HH, UK, 2.EaStCHEM School of Chemistry, The University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK and 3.Collaborative Optical Spectroscopy Micromanipulation and Imaging Centre (COSMIC), The University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK |
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| Abstract: | Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase. |
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