The RecBCD enzyme is a complex heterotrimeric helicase/nuclease that initiates recombination at double-stranded DNA breaks. In Escherichia coli, its activities are regulated by the octameric recombination hotspot, chi (5'-GCTGGTGG), which...
moreThe RecBCD enzyme is a complex heterotrimeric helicase/nuclease that initiates recombination at double-stranded DNA breaks. In Escherichia coli, its activities are regulated by the octameric recombination hotspot, chi (5'-GCTGGTGG), which is read as a single-stranded DNA sequence while the enzyme is unwinding DNA at over approximately 1,000 bp/s. Previous studies implicated the RecC subunit as the "chi-scanning element" in this process. Site-directed mutagenesis and phenotypic analyses identified residues in RecC responsible for chi recognition [Handa N, et al., (2012) Proc Natl Acad Sci USA, 10.1073/pnas.1206076109]. The genetic analyses revealed two classes of mutants. Here we use ensemble and single-molecule criteria to biochemically establish that one class of mutants (type 1) has lost the capacity to recognize chi (lost-recognition), whereas the second class (type 2) has a lowered specificity for recognition (relaxed-specificity). The relaxed-specificity mutants still recognize canonical chi, but they have gained the capacity to precociously recognize single-nucleotide variants of chi. Based on the RecBCD structure, these mutant classes define an alpha-helix responsible for chi recognition that is allosterically coupled to a structural latch. When opened, we propose that the latch permits access to an alternative exit channel for the single-stranded DNA downstream of chi, thereby avoiding degradation by the nuclease domain. These findings provide a unique perspective into the mechanism by which recognition of a single-stranded DNA sequence switches the translocating RecBCD from a destructive nuclease to a constructive component of recombinational DNA repair.
