1: Biochemistry. 1995 May 30;34(21):7127-34. 

Multiple amino acids determine the DNA binding specificity of the Msx-1
homeodomain.

Isaac VE, Sciavolino P, Abate C.

Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey
08854-5638, USA.

This study investigates the sequence features that contribute to the
differential DNA binding properties of two divergent homeodomains, Msx-1 and
HoxA3. We show that these homeodomains have overlapping, but nonidentical, DNA
binding site preferences. We defined the amino acid residues that contribute to
the observed differences in DNA binding specificity by producing a series of
mutated polypeptides in which selected residues in Msx-1 were replaced with the
corresponding ones in HoxA3. These analyses show that the DNA binding
specificity of Msx-1 versus HoxA3 results from the cumulative action of multiple
residues in all segments of the homeodomain (i.e., the N-terminal arm and
helices I, II, and III). Therefore, substitutions of residues in both helix III
and the N-terminal arm (but not in either segment alone) produced an Msx-1
polypeptide whose binding site preference was indistinguishable from that of
HoxA3. Residues in helices I and II also influence DNA binding activity; these
oppositely charged residues (e.g., lysine 19 and glutamate 30) may mediate ionic
interactions between helices I and II which stabilize DNA binding by Msx-1.
These findings demonstrate a critical interplay between residues in each
homeodomain segment for appropriate conformation of the protein-DNA complex.

PMID: 7766623 [PubMed - indexed for MEDLINE]


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2: Mol Cell Biol. 1993 Apr;13(4):2354-65. 

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity
of three murine homeodomain proteins.

Catron KM, Iler N, Abate C.

Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway,
New Jersey.

Murine homeobox genes play a fundamental role in directing embryogenesis by
controlling gene expression during development. The homeobox encodes a DNA
binding domain (the homeodomain) which presumably mediates interactions of
homeodomain proteins with specific DNA sites in the control regions of target
genes. However, the bases for these selective DNA-protein interactions are not
well defined. In this report, we have characterized the DNA binding
specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1.
We have identified optimal DNA binding sites for each of these proteins by using
a random oligonucleotide selection strategy. Comparison of the sequences of the
selected binding sites predicted a common consensus site that contained the
motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the
nucleotides flanking this core directed binding specificity. Whereas variations
in the nucleotides flanking the 5' side of the TAAT core produced modest
alterations in binding activity for all three proteins, perturbations of the
nucleotides directly 3' of the core distinguished the binding specificity of Hox
1.5 from those of Hox 7.1 and En-1. These differences in binding activity
reflected differences in the dissociation rates rather than the equilibrium
constants of the protein-DNA complexes. Differences in DNA binding specificities
observed in vitro may contribute to selective interactions of homeodomain
proteins with potential binding sites in the control regions of target genes.

PMID: 8096059 [PubMed - indexed for MEDLINE]


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