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Nucleic Acids Research, 2001, Vol. 29, No. 5 1054-1060
© 2001 Oxford University Press

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

Gerhard Wieland, Peter Hemmerich, Marianne Koch, Tanja Stoyan, Johannes Hegemann1 and Stephan Diekmann*

Institut für Molekulare Biotechnologie e.V., Beutenbergstrasse 11, D-07745 Jena, Germany and 1Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätstrasse 1, Geb. 26.12.01, D-40225 Düsseldorf, Germany

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.

* To whom correspondence should be addressed. Tel: +49 3641 656 260; Fax: +49 3641 656 261; Email: diekmann{at}imb-jena.de Present address: Tanja Stoyan, MCD Biology Department, University of California, Santa Barbara, CA 93106, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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