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 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 chi sites, are hotspots for RecBCD-promoted recombination and are 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 terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi-less DNA. Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi-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.
Kuzminov
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A
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Schabtach
E
E
Stahl
F W
FW
D001483:Base Sequence; D003360:Cosmids / Q000378:metabolism; D004249:DNA Damage; D004269:DNA, Bacterial / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011938:Rec A Recombinases; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C030894:terminase; D043211:Exodeoxyribonuclease V