X sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called χ sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-χ site interactions in vivo we used phage λ's terminase to introduce a site-specific double-strand break at λ's cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing χ sites in the proper orientation to the unprotected end is degraded more slowly in rec⁺ E.coli than is χ-less DNA. Increased survival of χ-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of χ-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.