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Molecular and Cellular Biology, December 2006, p. 8770-8780, Vol. 26, No. 23
0270-7306/06/$08.00+0     doi:10.1128/MCB.00949-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Binding of AlF-C, an Orc1-Binding Transcriptional Regulator, Enhances Replicator Activity of the Rat Aldolase B Origin{triangledown}

Hiroyuki Minami,1 Junko Takahashi,2,{dagger} Asami Suto,2 Yasushi Saitoh,1,2 and Ken-ichi Tsutsumi1,2*

Division of Functional Genomics, United Graduate School of Agricultural Sciences,1 Cryobiosystem Research Center, Iwate University, Morioka, Iwate 020-8550, Japan2

Received 30 May 2006/ Returned for modification 15 June 2006/ Accepted 6 September 2006

A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.

* Corresponding author. Mailing address: Cryobiosystem Research Center, Iwate University, Ueda, Morioka, Iwate 020-8550, Japan. Phone: 81 (0)19 621 6242. Fax: 81 (0)19 621 6242. E-mail: kentsu{at}iwate-u.ac.jp.

{triangledown} Published ahead of print on 18 September 2006.

{dagger} Present address: Research Center for Genomic Medicine, Saitama Medical School, Yamane, Hidaka, Saitama 350-1241, Japan.

Molecular and Cellular Biology, December 2006, p. 8770-8780, Vol. 26, No. 23
0270-7306/06/$08.00+0     doi:10.1128/MCB.00949-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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