An ORC Flip Enables Bidirectional Helicase Loading
Cells must duplicate their genetic material faithfully in every cell cycle to maintain cellular identity and ensure viability. The first step in DNA replication is the licensing of all potential origins by loading two inactive Mcm2-7 replicative DNA helicases around the origin DNA in a head-to-head...
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| Format: | Thesis |
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Massachusetts Institute of Technology
2022
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| Online Access: | https://hdl.handle.net/1721.1/145064 https://orcid.org/0000-0002-5446-7912 |
| Summary: | Cells must duplicate their genetic material faithfully in every cell cycle to maintain cellular identity and ensure viability. The first step in DNA replication is the licensing of all potential origins by loading two inactive Mcm2-7 replicative DNA helicases around the origin DNA in a head-to-head double hexamer. This conformation prepares the helicases to initiate bidirectional replication upon entry into S-phase. Interactions between three proteins and the Mcm2-7 helicase guide eukaryotic helicase loading. The origin-recognition complex (ORC) recognizes and binds origin DNA and recruits Cdc6. Mcm2-7 in complex with a third protein Cdt1 associates with ORC/Cdc6/DNA. Cdc6 and Cdt1 are sequentially released to allow loading of an oppositely-oriented second Cdt1-bound-Mcm2-7 hexamer that forms the Mcm2 7 double hexamer. Multiple mechanisms have been proposed to explain how two oppositely-oriented helicases are loaded at origins. Bulk in vitro helicase-loading experiments showed that two ORC binding sites are required, suggesting that two ORC proteins load the two Mcm2-7 helicases. Single-molecule observations, however, showed that a single ORC molecule can direct loading of both Mcm2-7 helicases.
In this thesis I present experiments that investigate interactions between ORC and Mcm2-7 to build a detailed model for ORC-guided helicase loading. Consistent with previous single-molecule studies, one DNA-bound ORC is sufficient for helicase loading. Each Mcm2-7 is recruited by an identical, short interaction with ORC that begins precisely when the corresponding Mcm2-7 arrives on DNA. Further, I detected and monitored a novel interaction of ORC with the initially recruited Mcm2 7 that was previously identified through structural studies. This interaction explains how ORC can be retained on the DNA during a binding-site transition or an “ORC flip”. The ORC flip consistently occurs between the two Mcm2 7 recruiting interactions, ensuring that the same ORC molecule recruits both Mcm2-7 molecules but in opposite orientations. Strikingly, the duration of the initial ORC-Mcm2-7 interaction coincides exactly with the presence of Cdt1 in the complex, suggesting Cdt1 release triggers ORC DNA release and flipping. Together these experiments reveal an intricate and carefully coordinated series of events that allow a single ORC to load two helicases in opposite orientations. |
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