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

Abstract

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.

We thank S. Fukushige (Tohoku University) for advice on Cre-lox recombination and for the generous gift of mouse recombinant L cell line 73-19 carrying a single copy of pSF73 DNA, S. Miyagawa (Osaka University) for the generous gift of an sCre recombinase gene optimized for expression in mammalian cells, and Bristol-Myers Squibb Pharma Company for providing a loxP targeting vector pBS226. We also thank M. Sakai and H. Ikeda (Hokkaido University) for valuable suggestions on the ChIP assay.

H.M. was a recipient of the student support program of COE to Iwate University from the Ministry of Education, Culture, Sports, Science and Technology of Japan.