CHI-SITES IN COMBINATION WITH RECA PROTEIN INCREASE THE SURVIVAL OF LINEAR DNA IN ESCHERICHIA-COLI BY INACTIVATING EXOV ACTIVITY OF RECBCD NUCLEASE

In Escherichia coil, unprotected linear DNA is degraded by exoV activi ty of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, c alled chi sites, are hotspots for RecBCD-promoted recombination and ar e shown, in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda'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 t erminase binding site. Linearized cosmid DNA containing chi sites in t he proper orientation to the unprotected end is degraded more slowly i n rec(+) E.coli than is chi-less DNA. Increased survival of chi-contai ning DNA is a result of partial inactivation of exoV activity and is d ependent on RecA and SSB proteins. The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structure s which have been proposed as intermediates in the RecBCD pathway of d ouble-strand break repair.