DNA double‐strand breaks caused by replication arrest

DNA double‐strand breaks caused by replication arrest We report here that DNA double‐strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse‐field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E.coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild‐type E.coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Wiley

DNA double‐strand breaks caused by replication arrest

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Publisher
Wiley
Copyright
Copyright © 2013 Wiley Periodicals, Inc
ISSN
0261-4189
eISSN
1460-2075
D.O.I.
10.1093/emboj/16.2.430
Publisher site
See Article on Publisher Site

Abstract

We report here that DNA double‐strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse‐field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E.coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild‐type E.coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression.

Journal

The EMBO JournalWiley

Published: Mar 15, 1998

Keywords: ; ; ; ;

References

  • When replication forks stop
    Bierne, H; Michel, B
  • Enzymatic DNA degradation in Escherichia coli: its relationship to synthetic processes at the chromosome level
    Buttin, G; Wright, M
  • Homologous genetic recombination: the pieces begin to fall into place
    Clark, AJ; Sandler, J
  • Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K‐12
    Gibson, FP; Leach, DRF; Lloyd, RG
  • In vivo studies of temperature‐sensitive recB and recC mutants
    Kushner, SR
  • Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme
    Lloyd, RG; Buckman, C; Benson, FE
  • A Short Course in Bacterial Genetics
    Miller, J
  • CHI and the RecBCD enzyme of Escherichia coli
    Myers, RS; Stahl, FW
  • Illegitimate recombination in mammalian cells
    Roth, D; Wilson, J
  • Differential suppression of priA2::kan phenotypes in Escherichia coli K12 by mutations in priA, lexA and dnaC
    Sandler, SJ; Samra, HS; Clark, AJ

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