TAU COUPLES THE LEADING-STRAND AND LAGGING-STRAND POLYMERASES AT THE ESCHERICHIA-COLI DNA-REPLICATION FORK

Synthesis of an Okazaki fragment occurs once every 1 or 2 s at the Esc herichia coli replication fork. To account for the rapid recycling req uired of the lagging-strand polymerase, it has been proposed that it i s held at the replication fork by protein-protein interactions with th e leading-strand polymerase as part of a dimeric polymerase assembly, Solution studies showed that the replicative polymerase, the DNA polym erase III holoenzyme, was indeed a dimer with two catalytic cores held together by the tau subunit. However, the functionality of this arran gement at the replication fork has never been demonstrated, We showed previously that the lagging-strand polymerase acted processively durin g multiple rounds of Okazaki fragment synthesis, i.e. the same polymer ase core assembly synthesized each and every fragment made by the fork . Using extreme dilution of active replication forks and the isolation of protein-DNA complexes capable of supporting coupled leading- and l agging-strand synthesis, we demonstrate here that this coupling of lea ding- and lagging-strand synthesis is, in fact, mediated by the tau su bunit of the holoenzyme acting as a physical bridge between the core a ssemblies synthesizing the leading and lagging strands.