Journal of Biological Chemistry
Volume 277, Issue 41, 11 October 2002, Pages 38416-38423
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DNA: REPLICATION REPAIR AND RECOMBINATION
The 14-3-3 Protein Homologues from Saccharomyces cerevisiae, Bmh1p and Bmh2p, Have Cruciform DNA-binding Activity and Associate in Vivo with ARS307*

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We have previously shown that, in human cells, cruciform DNA-binding activity is due to 14-3-3 proteins (Todd, A., Cossons, N., Aitken, A., Price, G. B., and Zannis-Hadjopoulos, M. (1998) Biochemistry37, 14317-14325). Here, wild-type and single- and double-knockout nuclear extracts from the 14-3-3 Saccharomyces cerevisiaehomologues Bmh1p and Bmh2p were analyzed for similar cruciform-binding activities in relation to these proteins. The Bmh1p-Bmh2p heterodimer, present in the wild-type strain, bound efficiently to cruciform-containing DNA in a structure-specific manner because cruciform DNA efficiently competed with the formation of the complex, whereas linear DNA did not. In contrast, the band-shift ability of the Bmh1p-Bmh1p and Bmh2p-Bmh2p homodimers present in thebmh2 and bmh1 single-knockout cells, respectively, was reduced by ∼93 and 82%, respectively. The 14-3-3 plant homologue GF14 was also able to bind to cruciform DNA, suggesting that cruciform-binding activity is a common feature of the family of 14-3-3 proteins across species. Bmh1p and Bmh2p were found to associate in vivo with the yeast autonomous replication sequence ARS307, as assayed by formaldehyde cross-linking, followed by immunoprecipitation with anti-Bmh1p/Bmh2p antibody and conventional PCR. In agreement with the finding of an association of Bmh1p and Bmh2p with ARS307, another immunoprecipitation experiment using 2D3, an anti-cruciform DNA monoclonal antibody, revealed the presence of cruciform-containing DNA in ARS307.

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Published, JBC Papers in Press, August 6, 2002, DOI 10.1074/jbc.M202050200

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This work was supported by the Canadian Institutes of Health Research (to M. Z.-H.) and the Cancer Research Society, Inc. (to M. Z.-H. and G. B. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.