Concerted interaction between origin recognition complex (ORC), nucleosomes and replication origin DNA ensures stable ORC-origin binding

Abstract

Chromosomal replication origins, where DNA replication is initiated, are determined in eukaryotic cells by specific binding of a six-subunit origin recognition complex (ORC). Many biochemical analyses have showed the detailed properties of the ORC-DNA interaction. However, because of the lack of in vitro analysis, the molecular architecture of the ORC-chromatin interaction is unclear. Recently, mainly from in vivo analyses, a role of chromatin in the ORC-origin interaction has been reported, including the existence of a specific pattern of nucleosome positioning around origins and of a specific interaction between chromatin-or core histones-and Orc1, a subunit of ORC. Therefore, to understand how ORC establishes its interaction with origin in vivo, it is essential to know the molecular mechanisms of the ORC-chromatin interaction. Here, we show that ORC purified from yeast binds more stably to origin-containing reconstituted chromatin than to naked DNA and forms a nucleosome-free region at origins. Molecular imaging using atomic force microscopy (AFM) shows that ORC associates with the adjacent nucleosomes and forms a larger complex. Moreover, stable binding of ORC to chromatin requires linker DNA. Thus, ORC establishes its interaction with origin by binding to both nucleosome-free origin DNA and neighboring nucleosomes. © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.