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14-3-3σ is a cruciform DNA binding protein and associates in vivo with origins of DNA replication

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Abstract

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms β, γ, ε, and ζ. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3σ isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3σ isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3σ antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3σ was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3σ antibody and quantitative PCR. The association of 14-3-3σ with the replication origins was maximal at the G1/S phase. The results indicate that 14-3-3σ is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding. J. Cell. Biochem. 87: 194–207, 2002. © 2002 Wiley-Liss, Inc.

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