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The origin recognition complex (ORC) is essential for initiation of eukaryotic chromosome replication as it loads the replicative helicase—the minichromosome maintenance (MCM) complex—at replication origins 1 . Replication origins display a stereotypic nucleosome organization with nucleosome depletion at ORC-binding sites and flanking arrays of regularly spaced nucleosomes 2 – 4 . However, how this nucleosome organization is established and whether this organization is required for replication remain unknown. Here, using genome-scale biochemical reconstitution with approximately 300 replication origins, we screened 17 purified chromatin factors from budding yeast and found that the ORC established nucleosome depletion over replication origins and flanking nucleosome arrays by orchestrating the chromatin remodellers INO80, ISW1a, ISW2 and Chd1. The functional importance of the nucleosome-organizing activity of the ORC was demonstrated by orc1 mutations that maintained classical MCM-loader activity but abrogated the array-generation activity of ORC. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, in addition to its canonical role as the MCM loader, has a second crucial function as a master regulator of nucleosome organization at the replication origin, a crucial prerequisite for efficient chromosome replication. Genome-scale in vitro reconstitution of DNA replication through chromatin establishes a crucial role for the origin recognition complex in organizing nucleosome arrays that are crucial for the initiation of replication.