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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Richardson TT,Wu X,Keith BJ,Heslop P,Jones AC,Connolly BA

doi

10.1093/nar/gks1364

subject

Has Abstract

pub_date

2013-02-01 00:00:00

pages

2466-78

issue

4

eissn

0305-1048

issn

1362-4962

pii

gks1364

journal_volume

41

pub_type

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