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.