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In Escherichia coli, several enzymes have been identified that participate in completing replication on the chromosome, including RecG, SbcCD, ExoI, and RecBCD. However, other enzymes are likely to be involved and the precise enzymatic mechanism by which this reaction occurs remains unknown. Two steps predicted to be necessary to complete replication are removal of Okazaki RNA fragments and ligation of the nascent strands at convergent replication forks. E. coli encodes two RNases that remove RNA-DNA hybrids, rnhA and rnhB, as well as two ligases, ligA and ligB. Here, we used replication profiling to show that rnhA and ligA, encoding RNase HI and Ligase A, participate in the completion reaction. Deletion of rnhA impaired the ability to complete replication and resulted in over-replication in the terminus region. It additionally suppressed initiation events from oriC, suggesting a role for the enzyme in oriC-dependent initiation, as has been suggested previously. We also show that a temperature-sensitive mutation in Ligase A led to over-replication at sites where replication completes, and that degradation at these sites occurred upon shifting to the nonpermissive temperature. Deletion of rnhB or ligB did not affect the growth or profile of replication on the genome.
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UV‐induced DNA damage disrupts the coordination between replication initiation, elongation and completion
Abstract Replication initiation, elongation and completion are tightly coordinated to ensure that all sequences replicate precisely once each generation. UV‐induced DNA damage disrupts replication and delays elongation, which may compromise this coordination leading to genome instability and cell death. Here, we profiled theEscherichia coligenome as it recovers from UV irradiation to determine how these replicational processes respond. We show thatoriCinitiations continue to occur, leading to copy number enrichments in this region. At late times, the combination of neworiCinitiations and delayed elongating forks converging in the terminus appear to stress or impair the completion reaction, leading to a transient over‐replication in this region of the chromosome. In mutants impaired for restoring elongation, includingrecA,recFanduvrA, the genome degrades or remains static, suggesting that cell death occurs early after replication is disrupted, leaving partially duplicated genomes. In mutants impaired for completing replication, includingrecBC,sbcCD xonAandrecG, the recovery of elongation and initiation leads to a bottleneck, where the nonterminus region of the genome is amplified and accumulates, indicating that a delayed cell death occurs in these mutants, likely resulting from mis‐segregation of unbalanced or unresolved chromosomes when cells divide.
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- Award ID(s):
- 1916625
- PAR ID:
- 10453446
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Genes to Cells
- Volume:
- 26
- Issue:
- 2
- ISSN:
- 1356-9597
- Page Range / eLocation ID:
- p. 94-108
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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