A role for 3′ exonucleases at the final stages of chromosome duplication in Escherichia coli

Chromosome duplication initiates via the assembly of replication fork complexes at defined origins, from where they proceed in opposite directions until they fuse with a converging fork. Recent work highlights that the completion of DNA replication is highly complex in both pro- and eukaryotic cells. In this study we have investigated how 3′ and 5′ exonucleases contribute towards the successful termination of chromosome duplication in Escherichia coli. We show that the absence of 3′ exonucleases can trigger levels of over-replication in the termination area robust enough to allow successful chromosome duplication in the absence of oriC firing. Over-replication is completely abolished if replication fork complexes are prevented from fusing by chromosome linearization. Our data strongly support the idea that 3′ flaps are generated as replication fork complexes fuse. In the absence of 3′ exonucleases, such as ExoI, these 3′ flaps can be converted into 5′ flaps, which are degraded by 5′ exonucleases, such as ExoVII and RecJ. Our data support the idea that multiple protein activities are required to process fork fusion intermediates. They highlight the complexity of fork fusions and further support the idea that the termination area evolved to contain fork fusion-mediated pathologies.