J. Biol. Chem., Vol. 255, Issue 22, 10671-10675, 11, 1980
Binding of the high mobility group protein, H6, to trout testis chromatin
L Kuehl, DJ Barton and GH Dixon
When 125I-labeled H6 was incubated with trout testis nuclei under
conditions of pH and ionic strength approximating those in vivo, the
radioactivity bound nearly quantitatively to the chromatin. Under
comparable conditions, most non-nuclear proteins do not bind. Binding was
neither tissue- nor species-specific, and HMG-17, a mammalian homolog of
H6, behaved similarly to H6. The labeled and the endogenous H6 molecules
were equivalent by several criteria: 1) Both were released nearly
quantitatively upon treatment of the chromatin with DNase I, whereas
neither was released by digestion with micrococcal nuclease, suggesting
that the labeled molecules, like those of endogenous H6, were bound
primarily to the core particles in transcriptionally competent portions of
the genome. 2) Salt extraction curves were similar for both the labeled and
unlabeled proteins, although about 15% of the labeled molecules were bound
to the chromatin more loosely than those of the endogenous H6. Taken
together, these results suggest that chromatin contains specific, well
defined sites to which H6 binds. Upon increasing the concentration of H6 in
the incubation mixture, progressively greater numbers of low affinity,
presumably nonspecific binding sites become occupied. This observation has
important implications for studies in which nucleases are employed to probe
chromatin structure, since they suggest that H6 molecules released from
specific, high affinity sites by the action of the nuclease might rebind
more loosely to other regions of the chromatin.
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