A helicase-tethered ORC flip enables bidirectional helicase loading

• Published in: eLife 2021-12, Vol.10
• Main Authors: Gupta, Shalini, Friedman, Larry J, Gelles, Jeff, Bell, Stephen P
• Format: Article
• Language: English
• Subjects: Binding Sites; Biochemistry and Chemical Biology; Cdt1; Cell cycle; Chromosomes and Gene Expression; Conformation; Deoxyribonucleic acid; DNA; DNA helicase; DNA replication; DNA Replication - genetics; DNA, Fungal - genetics; DNA, Fungal - metabolism; Experiments; Fluorescence resonance energy transfer; helicase; Mcm2-7; Microscopy; Minichromosome Maintenance Proteins - genetics; Minichromosome Maintenance Proteins - metabolism; ORC; origin licensing; Origin Recognition Complex - genetics; Origin Recognition Complex - metabolism; Protein Binding; Protein Domains; Protein Multimerization; Proteins; Recruitment; Replication Origin - genetics; Replication origins; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Spectroscopy
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/elife.74282

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 origin-recognition complex (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 single-molecule Förster resonance energy transfer (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.