Alteration of χ recognition by RecBCD reveals a regulated molecular latch and suggests a channel-bypass mechanism for biological control

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 23; pp. 8907 - 8912
• Main Authors: Yang, Liang, Handa, Naofumi, Liu, Bian, Dillingham, Mark S., Wigley, Dale B., Kowalczykowski, Stephen C.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 05-06-2012; National Acad Sciences
• Subjects: Biochemistry; Biological Sciences; DNA; DNA Repair - genetics; DNA Repair - physiology; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli Proteins - genetics; Exodeoxyribonuclease V - genetics; Exodeoxyribonuclease V - metabolism; Genetic mutation; Latches; Models, Molecular; Molecules; Mutagenesis, Site-Directed; Nucleotides; Protein Structure, Secondary - genetics; Protein Structure, Secondary - physiology; Regulatory sequences; Regulatory Sequences, Nucleic Acid - genetics; Substrate Specificity; Transcriptional regulatory elements
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1206081109

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, χ (5'-GCTGGTGG), which is read as a singlestranded DNA sequence while the enzyme is unwinding DNA at over ~1,000 bp/s. Previous studies implicated the RecC subunit as the "χ-scanning element" in this process. Site-directed mutagenesis and phenotypic analyses identified residues in RecC responsible for χ recognition [Handa N, et al., (2012) Proc Nati 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 χ (lost-recognition), whereas the second class (type 2) has a lowered specificity for recognition (relaxed-specificity). The relaxed-specificity mutants still recognize canonical χ, but they have gained the capacity to precociously recognize single-nucleotide variants of χ. Based on the RecBCD structure, these mutant classes define an α-helix responsible for χ 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 χ, 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.