Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.
The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication.
Alvarez
David
D
McGill University Department of Biochemistry and McGill Cancer Center Montreal, Canada H3G 1Y6.
Callejo
Mario
M
Shoucri
Rami
R
Boyer
Lee
L
Price
Gerald B
GB
Zannis-Hadjopoulos
Maria
M
D048948:14-3-3 Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D006801:Humans; D058619:Maltose-Binding Proteins; D009690:Nucleic Acid Conformation; D011233:Precipitin Tests; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C115090:cruciform-specific DNA-binding protein, human; D004247:DNA; D014446:Tyrosine 3-Monooxygenase