Identification of a Pax paired domain recognition sequence and evidence for DNA-dependent conformational changes.
Pax genes encode a family of developmentally regulated transcription factors that have been implicated in a number of human and murine congenital disorders, as well as in tumorigenesis (Gruss, P., and Walther, C. (1992) Cell 69, 719-722; Hill, R., and van Heyningen, V. (1992) Trends Genet. 8, 119-120; Chalepakis, G., Tremblay, P., and Gruss, P. (1992) J. Cell Sci. Suppl. 16, 61-67; Maulbecker, C. C., and Gruss, P. (1993) EMBO J. 12, 2361-2367; Walther, C., Guenet, J. L., Simon, D., Deutsch, U., Jostes, B., Goulding, M. D., Plachov, D., Balling, R., and Gruss, P. (1991) Genomics 11, 424-434; Barr, R. G., Galili, N., Holick, J., Biegel, J. A., Rovera, G., and Emanuel, B. S. (1993) Nature Genet. 3, 113-117). These genes are defined by the presence of an evolutionarily conserved DNA binding domain, termed the paired domain. The structure and the DNA binding characteristics of the paired domain remain largely unknown. We have utilized repetitive rounds of a polymerase chain reaction-based selection method to identify the optimal DNA binding sequences for the Pax-2 and Pax-6 paired domains. The results suggest that the paired domain family of peptides bind similar DNA sequences. Identification of this binding site has revealed an important structural clue regarding the mechanism of paired domain binding to DNA. CD and NMR structural analyses of the purified Pax-6 paired domain reveal it to be largely structureless in solution. Upon binding the recognition sequence, the complex becomes markedly less soluble and displays CD spectroscopic evidence of significant alpha-helical structure.
Epstein
J
J
Division of Cardiology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Cai
J
J
Glaser
T
T
Jepeal
L
L
Maas
R
R
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D016384:Consensus Sequence; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005136:Eye Proteins; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D051762:PAX2 Transcription Factor; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012097:Repressor Proteins; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D051762:PAX2 Transcription Factor; C497318:PAX2 protein, human; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C497319:Pax2 protein, mouse; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA