14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.
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 beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma 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. 14-3-3sigma 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-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.
Alvarez
David
D
Department of Biochemistry, McGill University, Montreal, Canada.
Novac
Olivia
O
Callejo
Mario
M
Ruiz
Marcia T
MT
Price
Gerald B
GB
Zannis-Hadjopoulos
Maria
M
D048948:14-3-3 Proteins; D000818:Animals; D014408:Biomarkers, Tumor; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002467:Cell Nucleus; D017931:DNA Primers / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005092:Exonucleases / Q000378:metabolism*; D005095:Exoribonucleases; D006367:HeLa Cells; D006801:Humans; D009363:Neoplasm Proteins; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000378:metabolism; D018741:Replication Origin / Q000502:physiology*
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D002457:Cell Extracts; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human