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Nucleic Acids Research, 2002, Vol. 30, No. 23 5205-5212
© 2002 Oxford University Press

Functional domains involved in the interaction between Orc1 and transcriptional repressor AlF-C that bind to an origin/promoter of the rat aldolase B gene

Yasushi Saitoh, Satoru Miyagi, Hiroyoshi Ariga1 and Ken-ichi Tsutsumi*

Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, 3-18-8, Ueda, Morioka, Iwate 020-8550, Japan and 1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan

*To whom correspondence should be addressed. Tel/Fax: +81 19 627 6242; Email: kentsu{at}iwate-u.ac.jp
Present address:
Satoru Miyagi, Division of Developmental Biology, Saitama Medical School, Research Center for Genomic Medicine, 1397-1. Yamane, Hidaka, Saitama 350-1241, Japan

The promoter of the rat aldolase B (AldB) gene functions in vivo as an origin of DNA replication in the cells in which transcription of the gene is repressed. Previously, we identified two closely related DNA-binding proteins, AlF-C1 and AlF-C2, which repressed the AldB gene promoter. We also reported that the binding site of these proteins, site C, is one of the required DNA elements of the AldB gene origin/promoter for autonomously replicating activity in transfected cells. In the present study, we show that AlF-C1 and AlF-C2 bind directly to Orc1, a subunit of the origin recognition complex (ORC). Deletion analyses revealed a functional domain in AlF-C2 for binding to Orc1, which is located separately from the DNA-binding domain. In addition, we found a novel protein-interacting domain in Orc1 required for the binding of AlF-C2, which was conserved in human, mouse and Chinese hamster, but not in Drosophila, frog and yeast. Thus, it is assumed that in mammalian cells, sequence- specific DNA-binding proteins are involved in recruiting ORC to regulate replication initiation and/or transcription repression.


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