title: Molecular determinants responsible for recognition of the single-stranded DNA regulatory sequence, χ, by RecBCD enzyme.
abstract: 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, χ (5'-GCTGGTGG-3'). Recognition of χ 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 χ 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 χ sequence. These mutants were characterized in vivo with regard to χ recognition, UV-sensitivity, phage degradation, and recombination proficiency. Of 38 residues mutated, 11 were previously undescribed mutations that altered χ recognition. The mutants fell into two classes: five that failed to respond to χ, and six that suggested a relaxed specificity for χ 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 χ recognition specificities.
authors.affiliation: Department of Microbiology, University of California, Davis, CA 95616, USA.
authors.forename: Naofumi
authors.initials: N
authors.lastname: Handa
authors.forename: Liang
authors.initials: L
authors.lastname: Yang
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dillingham
authors.forename: Ichizo
authors.initials: I
authors.lastname: Kobayashi
authors.forename: Dale B
authors.initials: DB
authors.lastname: Wigley
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Kowalczykowski
mesh_terms: D000595:Amino Acid Sequence; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; 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; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli