engleski

RecA730 dependent suppression of DNA repair deficiency in RecA loading mutants of Escherichia coli

Homologous recombination is essential for DNA repair and the maintenance of genome integrity. The most dangerous DNA damage are double-strand breaks (DSBs) which can arise during normal cell cycle or as a consequence of the action of DNA damaging agents. In Escherichia coli, repair of DSBs occurs by homologous recombination. The central part of recombination process is binding of RecA protein to ssDNA, i. e., production of RecA filament. DSBs can be processed into a RecA filament by the action of three enzymatic activities: helicase, 5’-3’exonuclease and RecA loading onto ssDNA. These activities are provided by the RecBCD enzyme in wild type cells or by the RecF pathway gene products in the recBC sbcBC(D) cells. The RecA730 mutant protein binds to ssDNA more efficiently than SSB protein, and consequently it is able to produce a RecA filament without the help of RecFOR mediators. We wanted to test whether the recA730 mutation can suppress DNA repair deficiency in derivatives of recB1080 mutant, i. e., in recB1080 recF(OR) and recB1080 recJ. The RecB1080CD enzyme has abolished nuclease and RecA loading activities, but retains helicase activity. In recB1080 mutant, the DSBs are repaired by the hybrid recombination pathway where helicase activity is provided by RecB1080CD enzyme, 5’-3’ exonuclease by RecJ protein and RecA loading by RecFOR complex. We looked on cell survival after γ-irradiation, and found that recA730 mutation suppresses DNA repair deficiency in recB1080 recF(OR) background where the defect is at the level of RecA loading, but not in recB1080 recJ background where the defect is at the level of nuclease activity.

RecA loading; RecA730; RecB1080CD; recombination

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