Premium
Proofreading dynamics of a processive DNA polymerase
Author(s) -
Ibarra Borja,
Chemla Yann R,
Plyasunov Sergey,
Smith Steven B,
Lázaro José M,
Salas Margarita,
Bustamante Carlos
Publication year - 2009
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/emboj.2009.219
Subject(s) - biology , proofreading , dna polymerase , polymerase , dna , dna clamp , genetics , dynamics (music) , computational biology , polymerase chain reaction , reverse transcriptase , gene , physics , acoustics
Replicative DNA polymerases present an intrinsic proofreading activity during which the DNA primer chain is transferred between the polymerization and exonuclease sites of the protein. The dynamics of this primer transfer reaction during active polymerization remain poorly understood. Here we describe a single‐molecule mechanical method to investigate the conformational dynamics of the intramolecular DNA primer transfer during the processive replicative activity of the Φ29 DNA polymerase and two of its mutants. We find that mechanical tension applied to a single polymerase–DNA complex promotes the intramolecular transfer of the primer in a similar way to the incorporation of a mismatched nucleotide. The primer transfer is achieved through two novel intermediates, one a tension‐sensitive and functional polymerization conformation and a second non‐active state that may work as a fidelity check point for the proofreading reaction.