Molecular determinants responsible for recognition of the single-stranded DNA regulatory sequence, χ, by RecBCD enzyme

The RecBCD enzyme is important for both restriction of foreign DNA and recombinational DNA repair. Switching enzyme function from the destructive antiviral state to the productive recombinational state is regulated by the recombination hotspot, chi (5'-GC-TGGTGG-3'). Recognition of chi is unique in that it is recognized as a specific sequence within single-stranded DNA (ssDNA) during DNA translocation and unwinding by RecBCD. The molecular determinants of chi recognition and the subsequent alteration in function are unknown. Consequently, we mutated residues within the RecC subunit that comprise a channel where ssDNA is thought to be scanned for a chi sequence. These mutants were characterized in vivo with regard to chi recognition, UV-sensitivity, phage degradation, and recombination proficiency. Of 38 residues mutated, 11 were previously undescribed mutations that altered chi recognition. The mutants fell into two classes: five that failed to respond to. chi, and six that suggested a relaxed specificity for chi recognition. The location of the first set of mutations defines a recognition structure responsible for sequence-specific binding of ssDNA. The second set defines a highly conserved structure, linked to the recognition structure, which we hypothesize regulates conversion of RecBCD from a molecular machine that destroys DNA to one that repairs it. These findings offer insight into the evolution of enzymes with alternate chi recognition specificities.