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Journal of Biochemistry Advance Access originally published online on March 18, 2009
Journal of Biochemistry 2009 146(1):77-85; doi:10.1093/jb/mvp048
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Influence of Magnesium Ion on the Binding of p53 DNA-Binding Domain to DNA-Response Elements

Yonglai Xue, Shuai Wang and Xizeng Feng*

College of Life Science, Nankai University, Tianjin, 300071, P. R. China

*To whom correspondence should be addressed. Tel: +86-22-2350-7022, Fax: +86-22-2350-7022, E-mail: xzfeng{at}nankai.edu.cn

Received January 14, 2009; Accepted March 2, 2009


  Abstract

Site-specific recognition and DNA-binding activity of p53 are crucial for its tumour suppressor function. Previous reports have shown that metal ions can affect the specific recognition and DNA-binding activity of p53DBD. Here we firstly report that magnesium ion can bind to the protein and influence its DNA-binding activity. To elucidate the nature and the effect of metal ions in the reaction chemistry, we utilized endogenous tryptophan fluorescence to quantitate the interaction between p53DBD and metal ions. The Ka value for the binding of Mg2+ to the protein is 1.88 x 103 M–1. Analysis of the CD data clearly suggested that the binding of magnesium ion induced a subtle conformational change rather than a radical modification of the overall protein architecture. Based on the results of electrophoretic mobility shift assays and fluorescence experiments, we concluded that the binding of Mg2+ significantly stimulated the binding of the protein to DNA in a sequence-independent manner, which differed from that of zinc ions in a sequence-specific manner. Based on these results and the fact that Mg2+ exists at relatively high concentration in the cell, we propose that Mg2+ is one of potential factors to affect or regulate the transactivation of p53.

Key Words: affinity, association constant, conformational change, DNA-binding activity, fluorescence, protein–DNA interaction

Abbreviations: CD, circular dichroism; DBD, DNA-binding domain; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; RGC, ribosomal gene cluster; SDS, sodium dodecyl sulfate; UV, ultraviolet


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