title: Collaborative coupling between polymerase and helicase for leading-strand synthesis.
abstract: Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between the helicase and the holoenzyme, consisting of the polymerase and trimeric clamp loaded by the clamp loader. We investigated the mechanism of this coupling on a DNA hairpin substrate manipulated by a magnetic trap. In stark contrast to the isolated enzymes, the coupled system synthesized DNA at the maximum rate without exhibiting fork regression or pauses. DNA synthesis and unwinding activities were coupled at low forces, but became uncoupled displaying separate activities at high forces or low dNTP concentration. We propose a collaborative model in which the helicase releases the fork regression pressure on the holoenzyme allowing it to adopt a processive polymerization conformation and the holoenzyme destabilizes the first few base pairs of the fork thereby increasing the efficiency of helicase unwinding. The model implies that both enzymes are localized at the fork, but does not require a specific interaction between them. The model quantitatively reproduces homologous and heterologous coupling results under various experimental conditions.
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: Vincent
authors.initials: V
authors.lastname: Croquette
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D017103:Bacillus Phages / Q000201:enzymology; D017122:Bacteriophage T4 / Q000201:enzymology; D004247:DNA / Q000096:biosynthesis*; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D020035:Holoenzymes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*; D000099272:Replisomes
chemical_list: D004247:DNA; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D020035:Holoenzymes; D009097:Multienzyme Complexes; D014764:Viral Proteins; D000099272:Replisomes; C063983:gene 41 protein, Enterobacteria phage T4; C069983:gene 43 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; C034970:bacteriophage T7 induced DNA polymerase