Conformational Dynamics of Single RecBCD Molecules

Abstract

RecBCD is a multifunctional enzyme possessing both helicase and nuclease activities. It harnesses the energy of ATP hydrolysis to processively unwind DNA. We used an optical-trapping assay featuring one base-pair stability to investigate the mechanism of RecBCD unwinding. Records of RecBCD motion at 6 pN of applied load showed fluctuations [4.1 ± 0.1 bp, (mean ± std. err.; freq. bandwidth = 0.1-10 Hz)] substantially above the control records with DNA alone. These fluctuations persisted when the enzyme's forward motion was stopped by removing ATP. Records of RecBCD bound to blunt-end DNA in the absence of ATP showed reduced dynamics (2.4 ± 0.2 bp), indicating the primary origin of the fluctuations was not due to anchoring via RecBCD. Prior biochemical studies showed that unwinding activity is preceded by an initiation phase consisting of several kinetic steps that generates a 10-nt, 5′-tailed substrate inside the RecBCD-DNA complex that engages RecD's helicase domain. This work also showed that binding to a forked 3′-(dT)6 and 5′-(dT)6 DNA substrate is kinetically equivalent to binding to a blunt-end DNA, while a 3′-(dT)6 and 5′-(dT)10substrate bypasses initiation. We found that records of RecBCD bound to these tailed DNA substrates showed fluctuations that quantitatively mirrored our records of RecBCD bound to blunt-end DNA and stopped within a long DNA substrate, respectively. Thus, the onset of large fluctuations in the RecBCD-DNA complex was coincident with that of unwinding activity. The magnitude and frequency of fluctuations increased when the DNA sequence immediately in front of the forked substrate was changed from GC to AT base pairs, consistent with RecBCD transiently translocating along the DNA without ATP hydrolysis. A tightly bound state with reduced dynamics (2.7 ± 0.1 bp) was observed with ADP-BeF2. These findings support a ratchet model for RecBCD movement.