title: Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil.
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.
authors.affiliation: Institute of Cell and Molecular Biosciences (ICaMB), The University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Tomas T
authors.initials: TT
authors.lastname: Richardson
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Wu
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Keith
authors.forename: Pauline
authors.initials: P
authors.lastname: Heslop
authors.forename: Anita C
authors.initials: AC
authors.lastname: Jones
authors.forename: Bernard A
authors.initials: BA
authors.lastname: Connolly
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005453:Fluorescence; D020027:Pyrococcus furiosus / Q000201:enzymology; D013698:Templates, Genetic; D014443:Tyrosine / Q000737:chemistry; D014498:Uracil / Q000737:chemistry*
chemical_list: D019843:Archaeal Proteins; D017931:DNA Primers; D014443:Tyrosine; D015075:2-Aminopurine; D014498:Uracil; D004247:DNA; D001120:Arginine; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases