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Binding of THZif-1, a MAZ-like zinc finger protein to the nuclease-hypersensitive element in the promoter region of the c-MYC protooncogene



Binding of THZif-1, a MAZ-like zinc finger protein to the nuclease-hypersensitive element in the promoter region of the c-MYC protooncogene



Journal of Biological Chemistry 271(49): 31322-31333



A detailed analysis is reported of the binding of the zinc finger protein THZif-1 to the nuclease-hypersensitive element (NHE) in the promoter region of the c-MYC gene using the electrophoretic mobility shift assay and a series of mutants of a fusion protein composed of glutathione S-transferase and THZif-1. The THZif-1 protein bound specifically to the single-stranded (ss) pyrimidine-rich DNA of the NHE (ss c-myc NHE-C) with an apparent dissociation constant (K-d(app)) of 0.077 mu-M. By contrast, no binding to the single-stranded purine-rich DNA of the NHE (ss c-myc NHE-G) was detected. Moreover, the binding affinity of THif-1 protein was 2-fold higher for the single-stranded 5-methyl-2'-deoxycytidine derivative of NHE (ss c-myc NBE-me-5C) than for the unmethylated NHE. In the case of the binding of THZif-1 to methylated double-stranded (ds) NHE (ds c-myc NHEme-5CG), no significant binding to the DNA was observed. The decrease in binding to DNA of THZif-1 was significant in the case of mutated ds c-myc NHE, in which more than two sites of deoxycytidine residues were methylated. However, the binding affinity of THZif-1 protein for methylated and for unmethylated triple-helical DNA of the NHE was almost identical. Moreover, the domain of the THZif-1 protein that made the major contribution to binding to ss c-myc NHE-C or ss c-myc NHE-me-5C corresponded to the amino-terminal second zinc finger motif. Taken together, the results indicate that the THZif-1 protein exhibits preferential DNA-binding activity with ss c-myc NHE-C, ds c-myc NBE-CG, and ts c-myc NHE but not with ss c-myc NHE-G and ds c-myc NHE-me-5CG in vitro.

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Accession: 008223320

Download citation: RISBibTeXText

PMID: 8940139

DOI: 10.1074/jbc.271.49.31322


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