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

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. © 2013 hoggard et al.