Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD-DNA complexes.
Nucleic Acids Res
; 44(12): 5849-60, 2016 07 08.
Article
en En
| MEDLINE
| ID: mdl-27220465
ABSTRACT
RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 µM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (â¼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
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ADN Bacteriano
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Proteínas de Escherichia coli
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Exodesoxirribonucleasa V
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Escherichia coli
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2016
Tipo del documento:
Article
País de afiliación:
Estados Unidos