Replication origins are licensed by loading two Mcm2‑7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2‑7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2‑7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first and second helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2‑7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.
Source data for the single-molecule experiments is provided as "intervals" files that can be read and manipulated by the Matlab program imscroll, which is publicly available: https://github.com/gelles-brandeis/CoSMoS_Analysis. These data can also be read directly in Matlab.The source data are archived at: https://doi.org/10.5061/dryad.547d7wm8z
A Helicase-tethered ORC Flip Enables Bidirectional Helicase LoadingDryad Digital Repository, doi:10.5061/dryad.547d7wm8z.
- Stephen P Bell
- Stephen P Bell
- Jeff Gelles
- Shalini Gupta
- Stephen P Bell
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Bruce Stillman, Cold Spring Harbor Laboratory, United States
© 2021, Gupta et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Doublecortin (DCX) is a microtubule (MT)-associated protein that regulates MT structure and function during neuronal development and mutations in DCX lead to a spectrum of neurological disorders. The structural properties of MT-bound DCX that explain these disorders are incompletely determined. Here, we describe the molecular architecture of the DCX–MT complex through an integrative modeling approach that combines data from X-ray crystallography, cryo-electron microscopy, and a high-fidelity chemical crosslinking method. We demonstrate that DCX interacts with MTs through its N-terminal domain and induces a lattice-dependent self-association involving the C-terminal structured domain and its disordered tail, in a conformation that favors an open, domain-swapped state. The networked state can accommodate multiple different attachment points on the MT lattice, all of which orient the C-terminal tails away from the lattice. As numerous disease mutations cluster in the C-terminus, and regulatory phosphorylations cluster in its tail, our study shows that lattice-driven self-assembly is an important property of DCX.
Listeria monocytogenes uses respiration to sustain a risky fermentative lifestyle during infection.