A helicase-tethered ORC flip enables bidirectional helicase loading

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.

A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

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. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession 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.

Selectivity of Lewy body protein interactions along the aggregation pathway of α-synuclein

The aggregation of alpha-synuclein (α-SYN) follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson’s Disease. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies.

Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay.

The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinsons disease. However, multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify which species can be detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent but our results reveal that their seeding behaviour is very different compared to preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in seeding assays.