What is it about?
We investigated how Escherichia coli finishes copying its circular chromosome, focusing on exonucleases, enzymes that trim DNA ends. When combinations of the 3′ exonucleases ExoI, ExoVII and SbcCD were missing, DNA became over-replicated in the termination area, where the two replication forks normally meet. In strains where the replication fork trap was removed and replication–transcription conflicts were reduced, this extra DNA synthesis could support chromosome duplication even when normal initiation at oriC was blocked. Linearising the chromosome, which prevents replication forks from fusing, abolished both the over-replication and this origin-independent growth. Genetic tests with PriA and RecJ support a model in which fork fusion can create short single-stranded DNA tails, or 3′ flaps, that are normally removed directly or converted into 5′ flaps for further processing.
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Why is it important?
Successful chromosome duplication depends not only on starting replication, but also on finishing it cleanly. Our study suggests that fork fusion in E. coli can generate DNA structures that need to be actively processed to prevent replication restarting in the termination area. This helps explain why the termination region and replication fork trap may act as a containment zone for problems created during the final stages of DNA replication.
Perspectives
What stands out to us is that a defect in DNA-end trimming produced enough termination-area synthesis to sustain replication without oriC firing under defined experimental conditions. By combining growth assays, marker-frequency sequencing, chromosome linearisation and targeted PriA/RecJ tests, we could connect a chromosome-level effect with a molecular model for fork-fusion intermediates. We also found it informative that the exonuclease pathway overlaps with, but is not identical to, the RecG pathway, highlighting that replication termination is more complex than a simple meeting of two forks.
Dr. Christian J Rudolph
Brunel University
Read the Original
This page is a summary of: A role for 3′ exonucleases at the final stages of chromosome duplication in Escherichia coli, Nucleic Acids Research, December 2018, Oxford University Press (OUP),
DOI: 10.1093/nar/gky1253.
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