title: Mechanism of strand displacement synthesis by DNA replicative polymerases.
abstract: Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.
authors.affiliation: Département de Physique, Laboratoire de Physique Statistique, Ecole Normale Supérieure, Université Pierre et Marie Curie Université Paris 06, Université Paris Diderot, Centre National de la Recherche Scientifique, Paris, 75005, France.
authors.forename: Maria
authors.initials: M
authors.lastname: Manosas
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Spiering
authors.forename: Fangyuan
authors.initials: F
authors.lastname: Ding
authors.forename: David
authors.initials: D
authors.lastname: Bensimon
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Allemand
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
authors.forename: Vincent
authors.initials: V
authors.lastname: Croquette
mesh_terms: D004247:DNA / Q000096:biosynthesis* / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D020035:Holoenzymes / Q000378:metabolism; D007700:Kinetics; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D020035:Holoenzymes; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase