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. 2001 Mar 1;29(5):1054-60.
doi: 10.1093/nar/29.5.1054.

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae

G Wieland  1 , P Hemmerich, M Koch, T Stoyan, J Hegemann, S Diekmann
Affiliations

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae

G Wieland et al. Nucleic Acids Res. .

Abstract

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.

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Figures

Figure 1
Figure 1
Expression of Cbf1 proteins. (A) Schematic drawing of the two proteins (His)6-Cbf1p and (His)6-Cbf1ΔN209p. The full-length protein is 351 amino acids in length while the deletion variant only contains the C-terminal amino acids 209–351 carrying the bHLHzip DNA binding motif. (B) Denaturing SDS–polyacrylamide gel of (His)6-Cbf1p (lane 1) and (His)6-Cbf1ΔN209p (lane 2). M, marker proteins with molecular weight indicated (kDa).
Figure 2
Figure 2
Binding of Cbf1p and Cbf1ΔN209p to CEN DNA. (A) Cbf1p binding. Lanes 1–15, increasing Cbf1p concentrations: lane 1, 0.63 nM; lane 2, 0.89 nM; lane 3, 1.04 nM; lane 4, 1.25 nM; lane 5, 1.39 nM; lane 6, 1.56 nM; lane 8, 1.79 nM; lane 9, 2.08 nM; lane 10, 2.50 nM; lane 11, 3.13 nM; lane 12, 4.17 nM; lane 13, 6.25 nM; lane 14, 12.5 nM; lane 15, 25 nM. (B) Cbf1ΔN209p binding. Lanes 1–15, increasing Cbf1ΔN209p concentrations: lane 1, 1.41 nM; lane 2, 2.01 nM; lane 3, 2.35 nM; lane 4, 2.82 nM; lane 5, 3.13 nM; lane 6, 3.52 nM; lane 8, 4.02 nM; lane 9, 4.69 nM; lane 10, 5.63 nM; lane 11, 7.05 nM; lane 12, 9.37 nM; lane 13, 14.07 nM; lane 14, 28.13 nM; lane 15, 56.25 nM. M, marker DNA in steps of 1 kb; F, free CEN DNA; C, complexed CEN DNA.
Figure 3
Figure 3
Binding analysis of Cbf1p and Cbf1ΔN209p to CEN15 DNA. The plot displays the data of gels shown in Figure 2. Triangles, binding values for Cbf1p; squares, binding values for Cbf1ΔN209p.
Figure 4
Figure 4
PAGE migration behavior of Cbf1p–CEN complexes. (A) All 16 CEN DNAs were incubated with Cbf1ΔN209p and analysed by native PAGE. (B) The migration length of the free CEN DNA was divided by the migration length of the respective Cbf1ΔN209p–CEN complex and the values are plotted for each CEN DNA. The data were derived from three independent experiments and represent mean values. Standard deviation was <5%.
Figure 5
Figure 5
p64-induced stimulation of Cbf1p DNA binding. (A) Coomassie stained SDS–gel of purified, recombinant CBF3 subunit p64 (lane 1). M, molecular weight standard proteins. (B) CEN 9 DNA was incubated with 10, 20 and 40 nM p64, and reaction mixtures analysed by gel shift (lanes 1, 2 and 3, respectively). No complex is observed. (C) CEN 9 DNA was incubated with 0.2 nM of Cbf1p (lanes 1 and 2) or Cbf1ΔN209p (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lane 2 and 4) of 20 nM p64.
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