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.
Highlights
Eukaryotic DNA replication initiates at specific chromosomal sites called origins
An origin is selected in G1-phase by the origin recognition complex (ORC) that directly binds chromosomal DNA, triggering a series of molecular events that culminate in the loading of an MCM helicase complex onto DNA
Multiple origins are required for accurate, efficient duplication—an insufficient number leads to mistakes in the genetic material and pathologies such as cancer
Summary
Eukaryotic DNA replication initiates at specific chromosomal sites called origins. An origin is selected in G1-phase by the origin recognition complex (ORC) that directly binds chromosomal DNA, triggering a series of molecular events that culminate in the loading of an MCM helicase complex onto DNA (reviewed in [1,2,3,4]). Temporal separation of the origin selection and activation steps helps ensure a chromosome is replicated only once per cell cycle (reviewed in [8]). It is unclear how the specific molecular events essential for the first step, in particular origin binding by ORC in G1 phase, might regulate the second step, origin activation in Sphase. Despite important advances, including several reported in recent studies, the specific molecular features of DNA replication origins that control their activation time remain incompletely understood [22,23,24,25,26,27]
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