Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Aug 11;22(15):3131–3137. doi: 10.1093/nar/22.15.3131

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains.

G Chalepakis 1, J Wijnholds 1, P Gruss 1
PMCID: PMC310286  PMID: 8065927

Abstract

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.

Full text

PDF
3131

Images in this article

3133Image
on p.3133
3134Image
on p.3134
3135Image
on p.3135
3135Image
on p.3135

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams B., Dörfler P., Aguzzi A., Kozmik Z., Urbánek P., Maurer-Fogy I., Busslinger M. Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis. Genes Dev. 1992 Sep;6(9):1589–1607. doi: 10.1101/gad.6.9.1589. [DOI] [PubMed] [Google Scholar]
  2. Baldwin C. T., Hoth C. F., Amos J. A., da-Silva E. O., Milunsky A. An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome. Nature. 1992 Feb 13;355(6361):637–638. doi: 10.1038/355637a0. [DOI] [PubMed] [Google Scholar]
  3. Balling R., Deutsch U., Gruss P. undulated, a mutation affecting the development of the mouse skeleton, has a point mutation in the paired box of Pax 1. Cell. 1988 Nov 4;55(3):531–535. doi: 10.1016/0092-8674(88)90039-6. [DOI] [PubMed] [Google Scholar]
  4. Balling R., Lau C. F., Dietrich S., Wallin J., Gruss P. Development of the skeletal system. Ciba Found Symp. 1992;165:132–143. doi: 10.1002/9780470514221.ch8. [DOI] [PubMed] [Google Scholar]
  5. Barr F. G., Galili N., Holick J., Biegel J. A., Rovera G., Emanuel B. S. Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nat Genet. 1993 Feb;3(2):113–117. doi: 10.1038/ng0293-113. [DOI] [PubMed] [Google Scholar]
  6. Bopp D., Burri M., Baumgartner S., Frigerio G., Noll M. Conservation of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila. Cell. 1986 Dec 26;47(6):1033–1040. doi: 10.1016/0092-8674(86)90818-4. [DOI] [PubMed] [Google Scholar]
  7. Burri M., Tromvoukis Y., Bopp D., Frigerio G., Noll M. Conservation of the paired domain in metazoans and its structure in three isolated human genes. EMBO J. 1989 Apr;8(4):1183–1190. doi: 10.1002/j.1460-2075.1989.tb03490.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chalepakis G., Fritsch R., Fickenscher H., Deutsch U., Goulding M., Gruss P. The molecular basis of the undulated/Pax-1 mutation. Cell. 1991 Sep 6;66(5):873–884. doi: 10.1016/0092-8674(91)90434-z. [DOI] [PubMed] [Google Scholar]
  9. Chalepakis G., Goulding M., Read A., Strachan T., Gruss P. Molecular basis of splotch and Waardenburg Pax-3 mutations. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3685–3689. doi: 10.1073/pnas.91.9.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chalepakis G., Stoykova A., Wijnholds J., Tremblay P., Gruss P. Pax: gene regulators in the developing nervous system. J Neurobiol. 1993 Oct;24(10):1367–1384. doi: 10.1002/neu.480241009. [DOI] [PubMed] [Google Scholar]
  11. Chalepakis G., Tremblay P., Gruss P. Pax genes, mutants and molecular function. J Cell Sci Suppl. 1992;16:61–67. doi: 10.1242/jcs.1992.supplement_16.8. [DOI] [PubMed] [Google Scholar]
  12. Crothers D. M., Gartenberg M. R., Shrader T. E. DNA bending in protein-DNA complexes. Methods Enzymol. 1991;208:118–146. doi: 10.1016/0076-6879(91)08011-6. [DOI] [PubMed] [Google Scholar]
  13. Czerny T., Schaffner G., Busslinger M. DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev. 1993 Oct;7(10):2048–2061. doi: 10.1101/gad.7.10.2048. [DOI] [PubMed] [Google Scholar]
  14. Dressler G. R., Douglass E. C. Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1179–1183. doi: 10.1073/pnas.89.4.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Epstein D. J., Malo D., Vekemans M., Gros P. Molecular characterization of a deletion encompassing the splotch mutation on mouse chromosome 1. Genomics. 1991 May;10(1):89–93. doi: 10.1016/0888-7543(91)90488-z. [DOI] [PubMed] [Google Scholar]
  16. Epstein D. J., Vekemans M., Gros P. Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3. Cell. 1991 Nov 15;67(4):767–774. doi: 10.1016/0092-8674(91)90071-6. [DOI] [PubMed] [Google Scholar]
  17. Epstein D. J., Vogan K. J., Trasler D. G., Gros P. A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch (Sp) mouse mutant. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):532–536. doi: 10.1073/pnas.90.2.532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fickenscher H. R., Chalepakis G., Gruss P. Murine Pax-2 protein is a sequence-specific trans-activator with expression in the genital system. DNA Cell Biol. 1993 Jun;12(5):381–391. doi: 10.1089/dna.1993.12.381. [DOI] [PubMed] [Google Scholar]
  19. Galili N., Davis R. J., Fredericks W. J., Mukhopadhyay S., Rauscher F. J., 3rd, Emanuel B. S., Rovera G., Barr F. G. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet. 1993 Nov;5(3):230–235. doi: 10.1038/ng1193-230. [DOI] [PubMed] [Google Scholar]
  20. Glaser T., Walton D. S., Maas R. L. Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet. 1992 Nov;2(3):232–239. doi: 10.1038/ng1192-232. [DOI] [PubMed] [Google Scholar]
  21. Goodman S. D., Nash H. A. Functional replacement of a protein-induced bend in a DNA recombination site. Nature. 1989 Sep 21;341(6239):251–254. doi: 10.1038/341251a0. [DOI] [PubMed] [Google Scholar]
  22. Goulding M. D., Chalepakis G., Deutsch U., Erselius J. R., Gruss P. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 1991 May;10(5):1135–1147. doi: 10.1002/j.1460-2075.1991.tb08054.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Goulding M., Sterrer S., Fleming J., Balling R., Nadeau J., Moore K. J., Brown S. D., Steel K. P., Gruss P. Analysis of the Pax-3 gene in the mouse mutant splotch. Genomics. 1993 Aug;17(2):355–363. doi: 10.1006/geno.1993.1332. [DOI] [PubMed] [Google Scholar]
  24. Gruss P., Walther C. Pax in development. Cell. 1992 May 29;69(5):719–722. doi: 10.1016/0092-8674(92)90281-g. [DOI] [PubMed] [Google Scholar]
  25. Hill R. E., Favor J., Hogan B. L., Ton C. C., Saunders G. F., Hanson I. M., Prosser J., Jordan T., Hastie N. D., van Heyningen V. Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature. 1991 Dec 19;354(6354):522–525. doi: 10.1038/354522a0. [DOI] [PubMed] [Google Scholar]
  26. Hoey T., Levine M. Divergent homeo box proteins recognize similar DNA sequences in Drosophila. Nature. 1988 Apr 28;332(6167):858–861. doi: 10.1038/332858a0. [DOI] [PubMed] [Google Scholar]
  27. Jordan T., Hanson I., Zaletayev D., Hodgson S., Prosser J., Seawright A., Hastie N., van Heyningen V. The human PAX6 gene is mutated in two patients with aniridia. Nat Genet. 1992 Aug;1(5):328–332. doi: 10.1038/ng0892-328. [DOI] [PubMed] [Google Scholar]
  28. Maulbecker C. C., Gruss P. The oncogenic potential of Pax genes. EMBO J. 1993 Jun;12(6):2361–2367. doi: 10.1002/j.1460-2075.1993.tb05890.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Morell R., Friedman T. B., Moeljopawiro S., Hartono, Soewito, Asher J. H., Jr A frameshift mutation in the HuP2 paired domain of the probable human homolog of murine Pax-3 is responsible for Waardenburg syndrome type 1 in an Indonesian family. Hum Mol Genet. 1992 Jul;1(4):243–247. doi: 10.1093/hmg/1.4.243. [DOI] [PubMed] [Google Scholar]
  30. Rojo F., Salas M. A DNA curvature can substitute phage phi 29 regulatory protein p4 when acting as a transcriptional repressor. EMBO J. 1991 Nov;10(11):3429–3438. doi: 10.1002/j.1460-2075.1991.tb04907.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tassabehji M., Read A. P., Newton V. E., Harris R., Balling R., Gruss P., Strachan T. Waardenburg's syndrome patients have mutations in the human homologue of the Pax-3 paired box gene. Nature. 1992 Feb 13;355(6361):635–636. doi: 10.1038/355635a0. [DOI] [PubMed] [Google Scholar]
  32. Tassabehji M., Read A. P., Newton V. E., Patton M., Gruss P., Harris R., Strachan T. Mutations in the PAX3 gene causing Waardenburg syndrome type 1 and type 2. Nat Genet. 1993 Jan;3(1):26–30. doi: 10.1038/ng0193-26. [DOI] [PubMed] [Google Scholar]
  33. Ton C. C., Hirvonen H., Miwa H., Weil M. M., Monaghan P., Jordan T., van Heyningen V., Hastie N. D., Meijers-Heijboer H., Drechsler M. Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region. Cell. 1991 Dec 20;67(6):1059–1074. doi: 10.1016/0092-8674(91)90284-6. [DOI] [PubMed] [Google Scholar]
  34. Treisman J., Gönczy P., Vashishtha M., Harris E., Desplan C. A single amino acid can determine the DNA binding specificity of homeodomain proteins. Cell. 1989 Nov 3;59(3):553–562. doi: 10.1016/0092-8674(89)90038-x. [DOI] [PubMed] [Google Scholar]
  35. Treisman J., Harris E., Desplan C. The paired box encodes a second DNA-binding domain in the paired homeo domain protein. Genes Dev. 1991 Apr;5(4):594–604. doi: 10.1101/gad.5.4.594. [DOI] [PubMed] [Google Scholar]
  36. Tsukamoto K., Tohma T., Ohta T., Yamakawa K., Fukushima Y., Nakamura Y., Niikawa N. Cloning and characterization of the inversion breakpoint at chromosome 2q35 in a patient with Waardenburg syndrome type I. Hum Mol Genet. 1992 Aug;1(5):315–317. doi: 10.1093/hmg/1.5.315. [DOI] [PubMed] [Google Scholar]
  37. Walther C., Guenet J. L., Simon D., Deutsch U., Jostes B., Goulding M. D., Plachov D., Balling R., Gruss P. Pax: a murine multigene family of paired box-containing genes. Genomics. 1991 Oct;11(2):424–434. doi: 10.1016/0888-7543(91)90151-4. [DOI] [PubMed] [Google Scholar]
  38. Wilson D., Sheng G., Lecuit T., Dostatni N., Desplan C. Cooperative dimerization of paired class homeo domains on DNA. Genes Dev. 1993 Nov;7(11):2120–2134. doi: 10.1101/gad.7.11.2120. [DOI] [PubMed] [Google Scholar]
  39. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  40. Zannini M., Francis-Lang H., Plachov D., Di Lauro R. Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters. Mol Cell Biol. 1992 Sep;12(9):4230–4241. doi: 10.1128/mcb.12.9.4230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. van der Vliet P. C., Verrijzer C. P. Bending of DNA by transcription factors. Bioessays. 1993 Jan;15(1):25–32. doi: 10.1002/bies.950150105. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES