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Fig. 2. Structural and functional analysis of the HOXA13 DNA-binding domain. (A)
The amino acid sequence of the A13-DBD peptide is presented above. (Left
panel) Structural analysis of the A13-DBD peptide by circular dichroism
spectroscopy indicates that the peptide folds into a stable 
-helical
DNA-binding motif at 4°C (blue curve). At 60°C, the majority of the
-helical content is lost by thermal denaturation (red curve). (Right
panel) Thermal stability measurements of the A13-DBD peptide showed that the
-helical conformation is maintained between 4 and 25°C. At
temperatures higher than 25°C, the peptide cooperatively transforms to its
denatured conformation. (B-E) A13-DBD peptide exhibits specific binding for
the DNA regions present upstream of Bmp2 and Bmp7. Asterisks
denote unbound radiolabeled DNA, arrowheads denote A13-DBD-DNA complexes
(B,C,E). (B) Quantitation of the A13-DBD affinity for the bound DNA regions
using increasing concentrations (black triangles) of cold competitor DNA (0,
150, 300 and 750 nM) revealed differential A13-DBD affinities for each of the
bound sites. (C) The Bmp2C2 region requires 2-fold greater
concentrations of A13-DBD to affect its electrophoretic mobility. (D)
Radiolabeled control DNA sequences exhibited no change in electrophoretic
mobility when incubated with 4-fold (2 µM) higher concentrations of the
A13-DBD peptide. (E) A13-DBD binding specificity was confirmed using the
Bmp7C1 binding site, which could not be displaced from the A13-DBD
peptide by using 750 nM concentrations of the unlabeled negative control DNA
(cNC), but is completely displaced using 750 nM unlabeled Bmp7C1
competitor DNA (cBMP7C1). (F) Analysis of the DNA sequences bound by the
A13-DBD reveals a novel series of HOX binding sites. The core TAAT site is
designated in bold type.
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