Assembly and activation of replicative helicases at origin DNA for replication initiation

To initiate DNA replication, it is essential to properly assemble a pair of replicative helicases at each replication origin. While the general principle of this process applies universally from prokaryotes to eukaryotes, the specific mechanisms governing origin selection, helicase loading, and subsequent helicase activation vary significantly across different species. Recent advancements in cryo-electron microscopy (cryo-EM) have revolutionized our ability to visualize large protein or protein-DNA complexes involved in the initiation of DNA replication. Complemented by real-time single-molecule analysis, the available high-resolution cryo-EM structures have greatly enhanced our understanding of the dynamic regulation of this process at origin DNA. This review primarily focuses on the latest structural discoveries that shed light on the key molecular machineries responsible for driving replication initiation, with a particular emphasis on the assembly of pre-replication complex (pre-RC) in eukaryotes.