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DNA nicks inflicted by restriction endonucleases are repaired by a RecA‐ and RecB‐dependent pathway in Escherichia coli
Author(s) -
Heitman Joseph,
Ivanenko Tatyana,
Kiss Antal
Publication year - 1999
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1999.01556.x
Subject(s) - ecorv , ecori , dna ligase , biology , recbcd , mutant , microbiology and biotechnology , dna , dna repair , escherichia coli , endonuclease , restriction enzyme , in vitro recombination , genetics , gene , molecular cloning , gene expression
Two mutants of the Eco RI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at Eco RI sites in vivo . Expression of the Eco RI endonuclease mutants in the absence of the Eco RI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56 , recB21 and lexA3 mutant strains of Escherichia coli . In parallel studies, overexpression of the Eco RV endonuclease in cells also expressing the Eco RV methyltransferase was used to introduce nicks at non‐cognate Eco RV sites in the bacterial genome. Eco RV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of Eco RV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.