A recent study delves into the mechanisms of DNA replication initiation in budding yeast, highlighting the importance of molecular intermediates in forming the double-hexamer (DH) complex essential for replication.

The research identifies the 'MO' intermediate, formed through ORC (origin recognition complex) binding, as critical for loading MCM helicases at low-affinity ORC binding sites.

Utilizing single-molecule experiments and cryo-electron microscopy, the study visualizes the dynamics of MCM loading and the stability of the helicase under various conditions, providing insights into the assembly and function of the replication machinery.

During the G1 phase of the cell cycle, MCM helicases are loaded at replication origins, transitioning to active CMG helicases during the S-phase, which initiate bidirectional DNA replication.

The research highlights that CDK phosphorylation of Orc2 inhibits MO formation, thus impacting the regulation of DNA replication by preventing MCM loading outside the G1 phase.

Orc2's intrinsically disordered region (IDR) plays a crucial role in MO formation, and mutations or CDK phosphorylation can disrupt this process, leading to reduced DH assembly at specific origins.

Experimental results indicate that while MO is essential for loading MCM at weak binding sites, it is not necessary when both binding sites are high-affinity, suggesting the existence of alternative loading mechanisms.