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14‐3‐3σ is a cruciform DNA binding protein and associates in vivo with origins of DNA replication
Author(s) -
Alvarez David,
Novac Olivia,
Callejo Mario,
Ruiz Marcia T.,
Price Gerald B.,
ZannisHadjopoulos Maria
Publication year - 2002
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.10294
Subject(s) - microbiology and biotechnology , cruciform , biology , origin recognition complex , dna replication , control of chromosome duplication , eukaryotic dna replication , chromatin immunoprecipitation , dna , s phase , immunoprecipitation , chromatin , western blot , replication factor c , antibody , biochemistry , gene , gene expression , genetics , promoter , archaeology , history
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 ors 8, 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 ors 8 and ors 12 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 G 1 /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.