Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae
AUTOR(ES)
Wieland, Gerhard
FONTE
Oxford University Press
RESUMO
Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix–loop–helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5′-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=29730Documentos Relacionados
- All 16 centromere DNAs from Saccharomyces cerevisiae show DNA curvature.
- Interaction of yeast kinetochore proteins with centromere–protein/transcription factor Cbf1
- Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1.
- Meiosis in Saccharomyces Cerevisiae Mutants Lacking the Centromere-Binding Protein Cp1
- Purification of a protein binding to the CDEI subregion of Saccharomyces cerevisiae centromere DNA.