Alteration of χ recognition by RecBCD reveals a regulated molecular latch and suggests a channel-bypass mechanism for biological control.
Proc Natl Acad Sci U S A
; 109(23): 8907-12, 2012 Jun 05.
Article
em En
| MEDLINE
| ID: mdl-22603793
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 â¼1,000 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA de Cadeia Simples
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Modelos Moleculares
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Sequências Reguladoras de Ácido Nucleico
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Exodesoxirribonuclease V
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Reparo do DNA
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Escherichia coli
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos