The C-terminal Subdomain Makes an Important Contribution to the DNA Binding Activity of the Pax-3 Paired Domain

Vogan, Kyle; Gros, Philippe

doi:10.1074/jbc.272.45.28289

Cited by 33 publications

(33 citation statements)

References 38 publications

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“…Although the N-terminal PAI has been noted in some cases to be the most critical subdomain, another study provides compelling evidence that the recognition of DNA targets by Pax proteins is achieved through the coordinate use of both subdomains. 19 Indeed, our demonstration of abrogation of DNA binding by a substitution mutation region within the C-subdomain supports the notion that this region plays a crucial role in the normal activities of the PAX9 paired domain. Our analysis of the paired domains of other members of the PAX gene family revealed another human genetic disorder caused by mutation of the equivalent Ile87 residue.…”

Section: Discussionsupporting

confidence: 64%

Molecular characterization of a novel PAX9 missense mutation causing posterior tooth agenesis

et al. 2006

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Autosomal dominant mutations in the gene encoding the paired box containing transcription factor PAX9 are associated with nonsyndromic human tooth agenesis that primarily affect posterior dentition. The molecular mechanisms contributing to its pathogenesis are poorly understood. In this study, we describe a novel mutation in PAX9 in a family with molar oligodontia. This heterozygous mutation results in the substitution of a highly conserved isoleucine residue by phenylalanine within the carboxyl-terminal subdomain of the paired domain. Immunolocalization and cell fractionation studies to ascertain the subcellular localization of the Ile87Phe protein showed that both wild-type and mutant proteins are synthesized in mammalian cells and that the mutation does not alter the nuclear localization of the mutant protein. Gel-shift assays using two cognate paired-domain recognition sequences, e5 and CD19-2(A-ins), revealed that while wild-type Pax9 binds to both sequences, the mutant protein was unable to bind these sites. In addition, the latter did not alter the DNA-binding activities of wild-type Pax9. Furthermore, we evaluated the ability of the Ile87Phe mutant protein to form a complex with a partner protein, Msx1, and found that the mutation under study has no effect on this interaction. Based on our observed defects in DNA binding by the mutant protein, we propose a loss-of-function mechanism that contributes to haploinsufficiency of PAX9 in this family with posterior tooth agenesis.

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Section: Discussionsupporting

confidence: 64%

Molecular characterization of a novel PAX9 missense mutation causing posterior tooth agenesis

et al. 2006

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“…The side chain carbonyl of residue 121 makes a water-mediated contact to base 19. The Ser-121 side chain may also make a similar contact as it has been shown that the C subdomain of Pax3 has DNA selectivity similar to that of Pax6 (Vogan and Gros 1997). Given the conservation of the C subdomain and the similar DNA-binding specificities of many paired domains, the Pax6 structure may provide a good basis for modeling DNA contacts by the C subdomain in other Pax proteins.…”

Section: Basis For Dna Recognition By the Pax C Subdomainmentioning

confidence: 97%

Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding

Xu¹,

Rould²,

Xu³

et al. 1999

Genes & Development

282

292

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Pax6, a transcription factor containing the bipartite paired DNA-binding domain, has critical roles in development of the eye, nose, pancreas, and central nervous system. The 2.5 Å structure of the human Pax6 paired domain with its optimal 26-bp site reveals extensive DNA contacts from the amino-terminal subdomain, the linker region, and the carboxy-terminal subdomain. The Pax6 structure not only confirms the docking arrangement of the amino-terminal subdomain as seen in cocrystals of the Drosophila Prd Pax protein, but also reveals some interesting differences in this region and helps explain the sequence specificity of paired domain-DNA recognition. In addition, this structure gives the first detailed information about how the paired linker region and carboxy-terminal subdomain contact DNA. The extended linker makes minor groove contacts over an 8-bp region, and the carboxy-terminal helix-turn-helix unit makes base contacts in the major groove. The structure and docking arrangement of the carboxy-terminal subdomain of Pax6 is remarkably similar to that of the amino-terminal subdomain, and there is an approximate twofold symmetry axis relating the polypeptide backbones of these two helix-turn-helix units. Our structure of the Pax6 paired domain-DNA complex provides a framework for understanding paired domain-DNA interactions, for analyzing mutations that map in the linker and carboxy-terminal regions of the paired domain, and for modeling protein-protein interactions of the Pax family proteins.

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“…Although this domain is not believed to play a critical role in DNA binding, current structural models suggest that it is in close proximity to DNA (25). In addition, alternative splicing of a glutamine residue at position 108 of Pax3/Pax7 is known to alter DNA binding specificity of the PD (54,58). Therefore, mutants Xa100 should be ideally suited to monitor structural changes associated with DNA binding by the PD.…”

Section: Discussionmentioning

confidence: 99%

Cross-talk between the Paired Domain and the Homeodomain of Pax3

Apuzzo

Abdelhakim

Fortin

et al. 2004

Journal of Biological Chemistry

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The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.Pax3 is a member of a family of 9 transcription factors (1), defined by a DNA binding module, the Paired domain (PD), 1 that was first identified in the Drosophila protein Paired (Prd) (2). Pax proteins play critical roles during normal embryonic development, and inactivating mutations cause major defects in development of skeleton, muscles, nervous system, eyes, kidneys, and the immune system (3-12). Pax3 is expressed in developing somites and in neural tube and neural crest cell derivatives and plays a role in the proliferation, migration, and differentiation of cells involved in neurogenesis and myogenesis (13, 14). The Pax3 mouse mutant (splotch, Sp) displays spina bifida and exencephaly and lacks limb muscles; likewise, mutations in human PAX3 cause Waardenburg syndrome (WS), a condition characterized by pigmentary disturbances, craniofacial abnormalities and sensorineuronal deafness (14 -21). PAX3 also plays a role in cell transformation as shown by the translocation t(2;13) (q35;q14) involving PAX3 (22) and forkhead-related transcription factor (23) associated with the solid tumor alveolar rhabdomyosarcoma. The translocation leads to the expression of a fusion protein containing the Ntermi...

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The C-terminal Subdomain Makes an Important Contribution to the DNA Binding Activity of the Pax-3 Paired Domain

Cited by 33 publications

References 38 publications

Molecular characterization of a novel PAX9 missense mutation causing posterior tooth agenesis

Molecular characterization of a novel PAX9 missense mutation causing posterior tooth agenesis

Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding

Cross-talk between the Paired Domain and the Homeodomain of Pax3

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