Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA

Abstract

ABSTRACTThe complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the ‘replication fork trap’ region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of Tus-ter and termination occurs when a converging replisome meets the non-permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the inclusion of lagging strand processing enzymes as well as several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.