Completion of DNA replication in Escherichia coli

Abstract

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.replication completion | double-strand break repair | RecBCD | homologous recombination | SbcDC

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

“…However, oriM2 has not been mapped accurately and it is not known whether the DSB that we observe relates to this origin. Several other results, such as the existence of terminal recombination (30)(31)(32) and the striking replication profile of a recB mutant (33), indicate that the terminus region of the chromosome presents an area of importance to recombination. However, there are very likely several different reactions taking place.…”

Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

“…Resequencing refers to comparing sequencing reads to an existing complete or draft genome. Whole-genome resequencing has a variety of applications in bacteriology, including identification of mutations in genetic suppressor screens (31), inferring genome replication status (32,33), and identifying mutations and structural variants in studies of genome instability (34). Despite the utility of resequencing, initial implementation of the various computational resources for alignment and variant calling can be challenging.…”

“…For many studies, it may be important to identify large structural variants in resequenced genomes, including large insertions, deletions, or rearrangements (33,34). Freebayes is able to detect indels with up to about half the sequenced read length; however, if a larger structural variation is to be detected, other tools must be employed.…”

“…Despite decades of research on replication, the process of termination was still unclear. A recent study used several approaches to show that the completion of DNA replication in E. coli involved the combined activities of DNA helicases and nucleases (33). In that study, changes in sequencing coverage, determined by resequencing, detected at the terminus region allowed Wendel et al to infer roles for RecG, RecBCD, SbcDC, and ExoI in resecting overreplicated DNA back to the completion point to help terminate DNA replication (33).…”

“…A recent study used several approaches to show that the completion of DNA replication in E. coli involved the combined activities of DNA helicases and nucleases (33). In that study, changes in sequencing coverage, determined by resequencing, detected at the terminus region allowed Wendel et al to infer roles for RecG, RecBCD, SbcDC, and ExoI in resecting overreplicated DNA back to the completion point to help terminate DNA replication (33). Analysis of sequencing coverage via whole-genome resequencing allowed the authors to find a new function for a very well-characterized group of enzymes (46).…”