title: Alteration of χ recognition by RecBCD reveals a regulated molecular latch and suggests a channel-bypass mechanism for biological control.
abstract: 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 single-stranded 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 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 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.
authors.affiliation: Department of Microbiology, University of California, Davis, CA 95616, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Yang
authors.forename: Naofumi
authors.initials: N
authors.lastname: Handa
authors.forename: Bian
authors.initials: B
authors.lastname: Liu
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dillingham
authors.forename: Dale B
authors.initials: DB
authors.lastname: Wigley
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Kowalczykowski
mesh_terms: D004260:DNA Repair / Q000235:genetics* / Q000502:physiology; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics; D043211:Exodeoxyribonuclease V / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary / Q000235:genetics / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli