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Nuclease activity is essential for RecBCD recombination in Escherichia coli
Author(s) -
Jockovich M. E.,
Myers R. S.
Publication year - 2001
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.1365-2958.2001.02573.x
Subject(s) - recbcd , helicase , nuclease , biology , exonuclease , recombinase , homologous recombination , site specific recombination , dna , mutant , dna repair , escherichia coli , recombination , microbiology and biotechnology , genetics , dna polymerase , gene , rna
RecBCD has two conflicting roles in Escherichia coli . (i) As ExoV, it is a potent double‐stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of χ recombination hot‐spots. These paradoxical roles are accommodated by χ‐dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that χ converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5′→ 3′ exonuclease activity is essential for recombination by RecBCD at χ recombination hot‐spots and at dsDNA ends in recD mutants. In the absence of RecB‐dependent nuclease function, recombination becomes entirely dependent on the 5′ → 3′ single‐stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).