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¹ Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
² Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan

The assembly of the prereplicative complex (pre-RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre-RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre-RC assembly was reconstituted in vitro using ARS1 DNA and purified origin-recognition complex (ORC), Cdc6p and Cdt1p-Mcm2-7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2-7p on to ARS1 DNA. When Mcm2-7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co-ordinately regulated during pre-RC assembly. In extracts from sid2-21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2-7p do not. However, Mcm2-7p does bind in the presence of exogenous Cdt1p or Cdt1p-Mcm2-7p complex. Cdt1p-Mcm2-7p complex, which was purified from G1-, early S or G2/M-arrested cells, exhibits structure-specific DNA binding, interacting only with bubble- or Y-shape-DNA, but the biological significance of this observation is not yet known.

Permanent address: ^aDepartment of Biotechnology and Bioinformatics, Chungbuk Provincial College of Science and Technology, 40 Geumgu-ri, Okcheon-eup, Okcheon-gun, Chungcheongbuk-do, 373-807, Republic of Korea

^bThese authors contributed equally to this work.

^* Correspondence: E-mail: ykawasak{at}fbs.osaka-u.ac.jp

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