The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.
- MeSH
- Escherichia coli genetika MeSH
- HCT116 buňky MeSH
- klonování DNA MeSH
- kompetitivní vazba MeSH
- křížová struktura DNA genetika metabolismus MeSH
- lidé MeSH
- plazmidy genetika MeSH
- proteiny 14-3-3 genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- replikace DNA genetika MeSH
- retardační test MeSH
- superhelikální DNA genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- adukty DNA genetika chemie MeSH
- cisplatina farmakologie chemie MeSH
- enzymy opravy DNA genetika chemie MeSH
- finanční podpora výzkumu jako téma MeSH
- oprava DNA imunologie MeSH
- superhelikální DNA genetika chemie MeSH
- techniky in vitro MeSH
- vazba proteinů genetika MeSH
- xeroderma pigmentosum genetika MeSH
- MeSH
- DNA vazebné proteiny biosyntéza genetika MeSH
- konformace nukleové kyseliny účinky léků MeSH
- krysa rodu rattus MeSH
- peptidové fragmenty biosyntéza genetika metabolismus MeSH
- proteiny s vysokou pohyblivostí genetika MeSH
- superhelikální DNA genetika metabolismus účinky léků MeSH
- transportní proteiny biosyntéza genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
