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J. Biol. Chem., Vol. 275, Issue 32, 24392-24399, August 11, 2000
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From the Department of Chemistry and Biochemistry, University of
California San Diego, La Jolla, California 92093
The DNA binding of three different NF-
Mechanism of
B DNA binding by Rel/NF-B dimers*
,
B
dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has
been examined using a combination of gel mobility shift and
fluorescence anisotropy assays. The NF-B p50/p65 heterodimer is
shown here to bind the B DNA target site of the immunoglobulin enhancer (Ig-B) with an affinity of approximately 10 nM. The p50 and p65 homodimers bind to the same site
with roughly 5- and 15-fold lower affinity, respectively. The nature of
the binding isotherms indicates a cooperative mode of binding for all
three dimers to the DNA targets. We have further characterized the role
of pH, salt, and temperature on the formation of the p50/p65
heterodimer-Ig-B complex. The heterodimer binds to the Ig-B DNA
target in a pH-dependent manner, with the highest affinity
between pH 7.0 and 7.5. A strong salt-dependent interaction
between Ig-B and the p50/p65 heterodimer is observed, with optimum
binding occurring at monovalent salt concentrations below 75 mM, with binding becoming virtually nonspecific at a salt
concentration of 200 mM. Binding of the heterodimer to DNA was unchanged across a temperature range between 4 °C and 42 °C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-B p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.
*
This work was supported in part by National Institutes of
Health Grant CA-71871 and fellowships from the Alfred P. Sloan and Hellman Foundations.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.
Supported by University of California San Diego Cellular and
Molecular Genetics Training Grant 2-T32-GM07240-24.
§
Supported by a predoctoral fellowship from the American Heart Association.
¶
To whom correspondence should be addressed. Tel.:
858-822-0469; Fax: 858-534-7042; E-mail:
gghosh@ucsd.edu.
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