2013/07/17

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

Division of Microbial Genetics • Araki Group

Concerted interaction between origin recognition complex (ORC), nucleosomes, and replication origin DNA ensures stable ORC–origin binding
Kohji Hizume, Masaru Yagura, and Hiroyuki Araki
Genes to Cells , 24 JUN 2013 DOI: 10.1111/gtc.12073. [Epub ahead of print]

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 revealed 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) reveals 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.

(1) DNA fragment containing origin-specific DNA motif (ACS).
(2) By chromatin reconstitution, nucleosome is formed on ACS.
(3) By the addition of ORC, ACS becomes nucleosome-free-region.
(4) ORC interacted to nucleosome-free ACS.
(5) ORC establishes its interaction with origin by binding to both nucleosome-free ACS and neighboring nucleosomes.


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