Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding

Abstract

The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5′-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5′-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5′-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3′-tail is encircled by the helicase while the displaced 5′-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing.

Dynamic look at DNA unwinding by a replicative helicase

“…Notably, "complete" unwinding, marked by an abrupt disappearance of fluorescence from the dye on the displaced DNA strand, was not observed, implying that an interaction between the helicase and the substrate persists following unwinding. This implies that the helicase does not dissociate from the DNA, possibly by interacting with the displaced strand on its outer surface, as was shown for Mini-Chromosome Maintenance (MCM) from Sulfolobus solfataricus (25,26). Strikingly, however, there was a high degree of heterogeneity in the unwinding patterns.…”

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

“…The number of smFRET states and transitions for Twinkle binding the excluded strand are much greater than that seen for SsoMCM or EcDnaB (24) 3 ; however, the dwell times in each state are similar. This implies that Twinkle does not necessarily have greater dynamics for binding the excluded strand; rather instead, it samples more exterior binding paths, especially for the 30/30 fork.…”

“…The 30/50 fork produced a nearly identical histogram, but the 30/40 substrate had larger low-and medium-FRET populations with a smaller high-FRET population. In the case of all three substrates, we see a significant high-FRET population, which has previously been shown to correspond to a wrapping interaction where the excluded strand wraps around the outer surface of the helicase (24,25). 3 The presence of multiple populations within the histogram data indicates that there are multiple FRET states sampled by each model fork substrate upon Twinkle binding.…”

“…8, A and B) or cyclo-dG (Fig. 8, C and D Twinkle Dynamically Interacts with Both DNA Strands-To test for a direct interaction between the helicase and the nontranslocating strand of the model DNA fork, single molecule FRET (smFRET) experiments were performed as described previously (24,25). Three FRET-labeled DNA fork substrates (30/30 (substrate 23), 40/30 (substrate 24), 50/30 (substrate 25)) were tested that had 30 bases (dT) on the 5Ј arm of the fork where the helicase loads, and increasing 30, 40, and 50 bases (dT) on the 3Ј excluded arm of the fork.…”

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase

“…Although DNA unwinding by helicases can be effectively measured in gel-based and fluorescence assays, the specific molecular interactions with DNA are poorly described. Previously, we have proposed that the excluded strand is not a passive component of unwinding; rather, it will interact along specific exterior paths on the hexameric helicase surface to both stabilize the complex and promote forward unwinding (1). Although structures of hexameric helicases that include small stretches of ssDNA have been solved recently (2,3), the interactions are constrained to central channel residues, and no molecular information is available at the duplex region or with respect to the excluded strand.…”