Mechanisms for Licensing Origins of DNA Replication in Eukaryotic Cells
The initiation of DNA replication in eukaryotic cells begins with the assembly of pre-Replicative Complexes (pre-RCs) at many sites along each chromosome during the G1 phase of the cell cycle. Pre-RCs license each chromosome for duplication during S phase and mark the origins of DNA replication. In this review, we discuss and contextualize recent findings elucidating the mechanisms of origin recognition and pre-RC assembly mediated by the Origin Recognition Complex (ORC), Cdc6 and the Mcm2-7 hexamer bound to Cdt1. We also present comprehensive movies that demonstrate the multiple mechanisms for pre-RC assembly and compare the structures of the complexes involved in human and S. cerevisiae cells.
Establishment of pre-RCs at the S. cerevisiae ARS1 origin
The process of establishment of pre-RCs at the S. cerevisiae ARS1 origin by a single ORC that cooperates with Cdc6 and Cdt1 to load two Mcm2-7 (MCM) hexamers in a head-to-head manner at each origin. A positioned nucleosome is bound to the DNA adjacent to the ARS1 origin. The A+B1 ORC binding site is relatively close to the inverted, but weaker ORC binding site B2. One ORC binds first to the A and B1 element and, with Cdc6 and Cdt1, loads the first MCM hexamer and then flips to the B2 element, where it cooperates with new Cdc6 and Cdt1 proteins to load a second MCM hexamer that then rotates to form the MCM double hexamer. The MCM hexamer bound to Cdt1 interacts with ORC-Cdc6 on DNA via winged helix domains in the MCM hexamer (initially in the Mcm3 and Mcm7 subunits of MCM (highlighted purple domains).
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Comparing yeast and mammalian ORC structures
A comparison of the structures of the S. cerevisiae ORC and the human ORC. The human ORC can exist in an auto-inhibited structure with the ORC1 subunit far away from the ORC4 subunit and the ORC2 winged helix in the channel that binds DNA. The ORC1 subunit rotates to interact with ORC4 to create an active ATP binding pocket and the ORC2 winged helix domain moves out of the DNA channel to allow ORC to bind DNA. The sources of the structures were PDB IDs 5ZR1 (yeast ORC), 5UJ7 (human ORC), 7JPO, 7JPP, 7JPQ, 7JPR, 7JPS (human ORC).
Pre-RC establishment by two Orcs
The process of establishment of pre-RCs at S. cerevisiae origins of DNA replication where the inverted B2 ORC binding site is further apart from the A+B1 element. In this case, the first MCM hexamer bound to Cdt1 is loaded by an ORC-Cdc6 complex and then the second MCM hexamer bound to Cdt1 is loaded by a second ORC-Cdc6 complex. The MCM hexamers rotate along the DNA, forming the MCM double hexamer.
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Comparing yeast and mammalian MCM double hexamers
Comparison of the S. cerevisiae MCM double hexamer and the human MCM double hexamer. The DNA in the S. cerevisiae double hexamer has duplex DNA pass through the MCM proteins in a zig-zag manner, but not unwound. The human MCM double hexamer is similar to the S. cerevisiae MCM double hexamer but the DNA in the human MCM double hexamer is unwound and the bases in the central base pair are completely separated. The sources of the structures were PDBs 5BK4 (yeast MCM double hexamer), 7W1Y (human MCM double hexamer).