In vitro helicase loading and ORC binding on replicative and non‐replicative ACSs

Abstract

ORC coordinates loading of the Mcm2–7 helicase complex, which leads to DNA replication. In the yeast S. cerevisiae, the origin recognition complex (ORC) recognizes a motif known as the ARS consensus sequence (ACS). Of the many ACS sites found in the yeast genome, only a small subset is actually bound by ORC. This correlates with the position of nucleosomes relative to the ACS: ChIP‐Seq analysis has shown that ORC only binds ACSs (called replicative (r) ACSs) that are within an asymmetric nucleosome free region (NFR), with precisely positioned adjacent nucleosomes and a downstream A‐rich region, most likely contributing to nucleosome exclusion. In contrast, ACSs where ORC does not bind (called non‐replicative (nr) ACSs) are located within smaller NFRs lacking the A‐rich region. We addressed the question whether nucleosome positioning dictates ORC binding and origin selection, and if sequence signatures are required in this process. Using an in vitro helicase loading assay, we showed that in the absence of nucleosomes, both rACSs and nrACSs could act as ORC binding sites and load the Mcm2–7 onto DNA. However, the rACSs load the helicase more efficiently than the nrACSs. Additionally, gel‐mobility shift assays showed that ORC has higher affinity for the rACSs. Our results indicate that sequences adjacent to the ACS play an important role in origin selection and function in the absence of nucleosomes.