Compact Origins and Where to Find Them: ORC's Guide to Genome-Wide Licensing

Abstract

Origin licensing is the first step in the fundamental process of DNA replication, which ensures the accurate transmission of an organism's genetic information. Studies in budding yeast have provided crucial insights into replication origins, revealing sequence-specific features and structural DNA elements guiding helicase loading. Here, we review the recent advances in our understanding of DNA replication origin licensing, focusing on insights into origin architecture and advancements in high-resolution sequencing. Progress in the field demonstrates that origins are compact units that load an individual MCM2-7 double hexamer, which in turn causes steric occlusion of the origin recognition complex (ORC) binding site. We discuss why, in addition to the DNA sequence, DNA shape, DNA flexibility, and correct spacing of A- and B2-elements are crucial for efficient helicase loading. These recent findings provide a mechanistic explanation for the regulation of genome-wide origin licensing and reveal fundamental principles of MCM2-7 helicase loading.

Keywords: ChIP‐Exo; DNA licensing; DNA replication; MCM2‐7; ORC; budding yeast; origins.

FIGURE 1

Budding yeast DNA licensing. (a) Simplified S. cerevisiae origin architecture. Origins typically consist of an A‐, B1‐, and B2‐element to load a stable MCM2‐7 double hexamer onto dsDNA. (b) ORC and Cdc6 form a complex on DNA and bend the DNA (PDB 7MCA [31]). (c) Cdt1‐MCM2‐7 becomes recruited, DNA is inserted, and the MCM2‐7 ring partially closes around DNA, resulting in the OCCM complex (PDB 5V8F [76]). Mcm4 ATP‐hydrolysis results in complete MCM2‐7 ring closure and Cdt1 and Cdc6 release. (d) ORC becomes repositioned to the other (N‐terminal) side of the MCM2‐7 hexamer, generating the MCM2‐7‐ORC complex (MO, PDB 6RQC [46]). (e) Following the recruitment of Cdc6, Cdt1‐MCM2‐7, and ATP‐hydrolysis‐dependent release of the helicase loading factors, the MCM2‐7 double‐hexamer forms around DNA (DH, PDB 5BK4 [50]).

FIGURE 2

Simplified budding yeast origin architecture. (a) Budding yeast replication origins contain conserved A‐, B1‐, and B2‐elements. The sequence motif of the A‐/B1‐element and the B2‐element are shown. The DNA sequences encode for specific DNA shapes, which support ORC‐dependent DNA bending. Frequently, the A‐/B1‐ and B2‐elements together take up 80 bp. (b) The A‐/B1‐ and B2‐elements can be found in varying distances, enriched for ∼12/13 bp intervals. An optimal distance is associated with optimal MCM2‐7 loading. We suggest that the ideal distance ensures the correct alignment of the two MCM2‐7 hexamers, where the Mcm3 N‐termini of each hexamer faces each other.

FIGURE 3

ChIP‐Exo in DNA replication research. (a) Schematic workflow of ChIP‐Exo sample preparation for next‐generation sequencing. Steps include formaldehyde cross‐linking, immunoprecipitation, on‐bead first adapter ligation, and digestion with exonuclease. This is followed by clean‐up, second adapter ligation (splint ligation), and library amplification. (b) Raw data comparison of various MCM2‐7 mapping techniques. MNase‐Seq [119], ChIP‐Seq [104], ChEC‐Seq [103], and ChIP‐Exo [82] traces are visualised in IGV (ver. 2.14.1, https://igv.org/) for ARS416 within a window of 3.5 kb). (c) ChIP‐Exo tag analysis (footprints) can distinguish between single and multiple, adjacent complexes. The strongest signal is found at the first protein‐DNA contact. Lambda exonuclease access is highlighted by black arrows.