Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil
| Autor: | Pauline Heslop, Tomas T. Richardson, Xiaohua Wu, Anita C. Jones, Brian J. Keith, Bernard A. Connolly |
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| Rok vydání: | 2013 |
| Předmět: |
Exonuclease
DNA polymerase Archaeal Proteins DNA-Directed DNA Polymerase Arginine Fluorescence 03 medical and health sciences chemistry.chemical_compound Genetics heterocyclic compounds 2-Aminopurine Uracil Polymerase DNA Primers 030304 developmental biology 0303 health sciences biology Nucleic Acid Enzymes 030302 biochemistry & molecular biology DNA Templates Genetic biology.organism_classification Pyrococcus furiosus Exodeoxyribonucleases chemistry Biochemistry Coding strand biology.protein Tyrosine Primer (molecular biology) |
| Zdroj: | Richardson, T T, Wu, X, Keith, B J, Heslop, P, Jones, A C & Connolly, B A 2013, ' Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil ', Nucleic Acids Research . https://doi.org/10.1093/nar/gks1364 Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | 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 0 –5 0 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 timeresolved 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 primertemplate strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase. |
| Databáze: | OpenAIRE |
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