Recruitment of Bacillus subtilis RecN to DNA Double-Strand Breaks in the Absence of DNA End Processing

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Abstract
The recognition and processing of double-strand breaks (DSBs) to a 3′ single-stranded DNA (ssDNA) overhang structure in Bacillus subtilis is poorly understood. Mutations in addA and addB or null mutations in recJ (Δ recJ ), recQ (Δ recQ ), or recS (Δ recS ) genes, when present in otherwise-Rec + cells, render cells moderately sensitive to the killing action of different DNA-damaging agents. Inactivation of a RecQ-like helicase (Δ recQ or Δ recS ) in addAB cells showed an additive effect; however, when Δ recJ was combined with addAB , a strong synergistic effect was observed with a survival rate similar to that of Δ recA cells. RecF was nonepistatic with RecJ or AddAB. After induction of DSBs, RecN-yellow fluorescent protein (YFP) foci were formed in addAB Δ recJ cells. AddAB and RecJ were required for the formation of a single RecN focus, because in their absence multiple RecN-YFP foci accumulated within the cells. Green fluorescent protein-RecA failed to form filamentous structures (termed threads) in addAB Δ recJ cells. We propose that RecN is one of the first recombination proteins detected as a discrete focus in live cells in response to DSBs and that either AddAB or RecQ(S)-RecJ are required for the generation of a duplex with a 3′-ssDNA tail needed for filament formation of RecA.