The packaging of eukaryotic DNA into nucleosomes is a critical regulator of
nuclear events. To address the interplay between chromatin and replication
initiation, we have assessed the determinants and function of the nucleoso
mal configuration of S. cerevisiae replication origins. Using in vitro and
in vivo assays, we demonstrate that the yeast initiator, the origin recogni
tion complex (ORC), is required to maintain the nucleosomal configuration a
djacent to origins. Disruption of the ORC-directed nucleosomal arrangement
at an origin interferes with initiation of replication, but does not alter
the association of ORC with the origin. Instead, the nucleosomes positioned
by ORC are important for prereplicative complex formation. These findings
suggest that origin-proximal nucleosomes facilitate replication initiation,
and that local chromatin structure affects origin function.