Nucleic Acids Research, Vol 26, Issue 20 4574-4581, Copyright © 1998 by Oxford University Press
AS Fortin, DA Underhill and P Gros
Pax3 contains two structurally independent DNA-binding domains, a paired
domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies
have shown that both domains are functionally interdependent. In
particular, it has been shown that the PD can regulate the DNA- binding
specificity and dimerization potential of the HD. To delineate Pax3 protein
segments that are involved in the regulation of HD DNA- binding, a series
of chimeric proteins were created in which the HD and linker region were
gradually replaced with corresponding sequences from a heterologous HD
protein, Phox. Characterization of chimeric proteins by electrophoretic
mobility shift analysis (EMSA) suggests that a portion of the linker region
contributes to the functional interaction between the PD and HD. In
addition, stepwise removal of sequences from the Pax3 PD was used to define
regions within this domain that are involved in the regulation of HD
DNA-binding. EMSA of these proteins in the context of the chimeric
Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain
of the PD does not play a major role in the regulation of HD DNA-binding
and (ii) the N-terminal subdomain and, in particular, the second
alpha-helix are essential for modulation of HD DNA-binding. Significantly,
deletion of helix 2 was found to be sufficient to uncouple regulation of HD
DNA-binding by the PD.
ARTICLES
Helix 2 of the paired domain plays a key role in the regulation of DNA- binding by the Pax-3 homeodomain
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
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