DNA DOUBLE-STRAND BREAKS CAUSED BY REPLICATION ARREST

We report here that DNA double-strand breaks (DSBs) form in Escherichi a coli upon arrest of replication forks due to a defect in, or the inh ibition of, replicative DNA helicases, The formation of DSBs was asses sed by the appearance of Linear DNA detected by pulse-held gel electro phoresis, Processing of DSBs by recombination repair or linear DNA deg radation was abolished by mutations in recBCD genes. Two E. coli repli cative helicases were tested, Rep, which is essential in recBC mutants , and DnaB. The proportion of linear DNA increased up to 50% upon shif t of rep recBTS recCTS cells to restrictive temperature, No increase i n linear DNA was observed in the absence of replicating chromosomes, i ndicating that the formation of DSBs in rep strains requires replicati on. Inhibition of the DnaB helicase either by a strong replication ter minator or by a dnaBTS mutation led to the formation of linear DNA, sh owing that blocked replication forks are prone to DSB formation, In wi ld-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 amoun t of spontaneous DSBs, We propose that some of them may also result fr om the impairment of replication fork progression.