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: Jan 15, 1997

References

  • When replication forks stop
    Bierne, Bierne; Michel, Michel
  • The mechanism of recA polA lethality: suppression by RecA‐independent recombination repair activated by the lexA (Def) mutation in Escherichia coli
    Cao, Cao; Kogoma, Kogoma
  • The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination
    Connelly, Connelly; Leach, Leach
  • In vivo biochemistry: physical monitoring of recombination induced by site‐specific endonuclease
    Haber, Haber
  • Collapse and repair of replication forks in Escherichia coli
    Kuzminov, Kuzminov
  • DNA helicases: enzymes with essential roles in all aspects of DNA metabolism
    Matson, Matson; Bean, Bean; George, George
  • CHI and the RecBCD enzyme of Escherichia coli
    Myers, Myers; Stahl, Stahl
  • Overlapping functions for recF and priA in cell viability and UV‐inducible SOS expression are distinguished by dnaC809 in Escherichia coli K‐12
    Sandler, Sandler
  • Insertion of inverted Ter sites into the terminus region of Escherichia coli chromosome delays completion of DNA replication and disrupts the cell cycle
    Sharma, Sharma; Hill, Hill
  • Lethality of rep recB and rep recC double mutants of Escherichia coli
    Uzest, Uzest; Ehrlich, Ehrlich; Michel, Michel

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