Proofreading dynamics of a processive DNA polymerase.
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 Phi 29 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.
Ibarra
Borja
B
Department of Physics, University of California, Berkeley, CA 94720-3220, USA.
Chemla
Yann R
YR
Plyasunov
Sergey
S
Smith
Steven B
SB
Lázaro
José M
JM
Salas
Margarita
M
Bustamante
Carlos
C
D001435:Bacteriophages / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D008968:Molecular Conformation; D009154:Mutation; D009690:Nucleic Acid Conformation; D052898:Optical Tweezers; D016133:Polymerase Chain Reaction; D011108:Polymers / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013314:Stress, Mechanical
D011108:Polymers; D004247:DNA; D004259:DNA-Directed DNA Polymerase