A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

Abstract

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.

Author Summary

Cell division requires the duplication of chromosomes, protein-DNA complexes harboring genetic information. Specific chromosomal positions, origins, initiate this duplication. Multiple origins are required for accurate, efficient duplication—an insufficient number leads to mistakes in the genetic material and pathologies such as cancer. Origins are chosen when the origin recognition complex (ORC) binds to them. The molecular interactions controlling this binding remain unclear. Understanding these interactions will lead to new ways to control cell division, which could aid in treatments of disease. Experiments were performed in the eukaryotic microbe budding yeast to define the types of molecular interactions ORC uses to bind origins. Yeasts are useful for these studies because chromosome duplication and structure are well conserved from yeast to humans. While ORC-DNA interactions were important, interactions between ORC and chromosomal proteins played a role. In addition, different origins relied on different types of molecular interactions with ORC. Finally, ORC-protein interactions but not ORC-DNA interactions were associated with enhanced origin function during chromosome-duplication, revealing an unanticipated link between the types of molecular interactions ORC uses to select an origin and the ultimate function of that origin. These results have implications for interfering with ORC-origin interactions to control cell division.