Completion of DNA replication in Escherichia coli

The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.During chromosomal replication, cells tightly regulate the processes of initiation, elongation, and completion to ensure that each daughter cell inherits an identical copy of the genetic information. Although the mechanisms regulating initiation and elongation have been well characterized (reviewed in refs. 1, 2), the process of how cells recognize replicated regions and complete replication at the precise doubling point remains a fundamental question yet to be addressed. Whether this event occurs once per generation as in Escherichia coli or thousands of times per generation as in human cells, the failure to efficiently carry out this function would be expected to result in a loss of genomic stability. Considering the large number of proteins that cells devote to ensuring the fidelity of replication initiation and elongation, it seems highly probable that the final critical step in this process will be also be tightly regulated and controlled enzymatically.In some aspects, one could argue that the efficiency of completion is likely to be more critical to the faithful duplication of the genome than that of initiation. When replication origins fail to initiate efficiently, elongation of replication forks from neighboring origins is often able to compensate (3, 4), and both prokaryotic and eukaryotic cells are able to tolerate variations in their origin number without severe phenotypic consequences (5–7). However, a failure to accurately limit or join any event where forks converge would be expected to result in duplications, deletions, rearrangements, or a loss of viability depending upon how the DNA ends are resolved at segregation.A number of studies suggest that an ability to sense when all sequences in the genome have doubled is critical to genomic replication. In vitro, converging replisomes continue through their meeting point as one replisome displaces the other, resulting in over-replication, or a third copy, of the region where the forks meet (8). Complicating the process of genomic doubling even further, several studies have suggested that illegitimate initiations of replication frequently occur at single-strand nicks, gaps, D-loops, and R-loops throughout the genomes of both prokaryotes and eukaryotes (9–14). Similar to when replication forks continue through a previously replicated template, each of these events would generate a third copy of the chromosomal region where the event occurs. Thus, over-replication may be inherent and promiscuous during the duplication of genomes. If true, then to ensure that each sequence of the genome replicates once, and only once, per generation, cells must encode an enzymatic system that is essentially able to count in pairs and efficiently degrade odd or over-replicated regions until the two nascent end pairs of replication events can be joined.The model organism E. coli is particularly well-suited to dissect how this fundamental process occurs. In E. coli, the completion of replication occurs at a defined region on the genome, opposite to the bidirectional origin of replication (15). Most completion events can be further localized to one of six termination (ter) sequences within the 400-kb terminus region due to the action of Tus, which binds to ter and inhibits replication fork progression in an orientation-dependent manner, in effect stalling the replication fork at this site until the second arrives (16, 17). Although Tus confines converging replication forks to a specific region, it does not appear to be directly involved in the completion reaction because tus mutants have no phenotype and complete replication normally (18). Furthermore, plasmids and bacteriophage lacking ter sequences are maintained stably (19).Many mutants impaired for either replication initiation or elongation were initially isolated based on their growth defects or an impaired ability to maintain plasmids (20–22). We reasoned that mutants impaired for the ability to complete replication might be expected to exhibit similar phenotypes and initially focused our attention on the properties of recBC and recD mutants. RecB-C-D forms a helicase–nuclease complex that is required for homologous repair of double-strand breaks in E. coli (23, 24). The enzyme uses specific DNA sequences, termed “Chi sites,” to initiate recombination between pairs of molecules. Loss of RecB or C inactivates the enzyme complex, whereas loss of RecD inactivates the nuclease and Chi recognition, but retains helicase activity (23, 24). Here, we show that inactivation of RecBCD leads to a failure to recognize and join replicating molecules at their doubling point. Although the completion process requires RecBCD, it is distinct from double-strand break repair and does not involve a double-strand break intermediate, homologous recombination, or RecA.