A helicase-tethered ORC flip enables bidirectional helicase loading
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Evaluation Summary:
The paper describes single-molecule experiments that address the assembly of a double hexamer of the Mcm2-7 complex that is required to license all origins of DNA replication in eukaryotic cells by formation of a pre-Replicative Complex (pre-RC). The observations show that one Origin Recognition Complex, an ATP-dependent DNA binding protein, can load both Mcm2-7 hexamers in opposite orientation. The results nicely complement prior data on the mechanism of pre-RC assembly.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
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.
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Evaluation Summary:
The paper describes single-molecule experiments that address the assembly of a double hexamer of the Mcm2-7 complex that is required to license all origins of DNA replication in eukaryotic cells by formation of a pre-Replicative Complex (pre-RC). The observations show that one Origin Recognition Complex, an ATP-dependent DNA binding protein, can load both Mcm2-7 hexamers in opposite orientation. The results nicely complement prior data on the mechanism of pre-RC assembly.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Joint Public Review:
Gupta et al. investigate the mechanism of Mcm2-7 helicase loading using an in vitro reconstituted S. cerevisiae system by single molecule colocalization spectroscopy and sm-FRET. Previous biochemical and single-molecule studies have led to contrasting models as to whether one or two ORCs are involved in recruiting and loading both Mcm2-7 hexamers in a double hexamer. How the transitions between OCCM and MO loading intermediates are coordinated was likewise unknown. Using COSMOS and sm-FRET, the authors convincingly show that a) a single ORC recruits both the first and second Mcm2-7 hexamer in the majority of observed loading events, b) ORC recruits the first and second Mcm2-7 hexamer using similar ORC-MCM interactions, c) ORC is retained at the origin by the formation of an MO complex and these interactions …
Joint Public Review:
Gupta et al. investigate the mechanism of Mcm2-7 helicase loading using an in vitro reconstituted S. cerevisiae system by single molecule colocalization spectroscopy and sm-FRET. Previous biochemical and single-molecule studies have led to contrasting models as to whether one or two ORCs are involved in recruiting and loading both Mcm2-7 hexamers in a double hexamer. How the transitions between OCCM and MO loading intermediates are coordinated was likewise unknown. Using COSMOS and sm-FRET, the authors convincingly show that a) a single ORC recruits both the first and second Mcm2-7 hexamer in the majority of observed loading events, b) ORC recruits the first and second Mcm2-7 hexamer using similar ORC-MCM interactions, c) ORC is retained at the origin by the formation of an MO complex and these interactions stabilize the first Mcm2-7 hexamer on DNA and d) Cdt1 release coordinates the transition between OCCM and MO complexes. These data are consistent with the proposed ORC-flip model, which posits that ORC is released from the original DNA site and rebinds on the opposite side of the first loaded Mcm2-7 for loading of the second Mcm2-7. This work provides important new insights into understanding the mechanisms of bidirectional replicative helicase loading.
The paper is an important contribution and the reviewers asked for a number of clarifications and explanations about the data.
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