An Arginine Residue Instead of a Conserved Leucine Residue in the Recognition Helix of the Finger 3 of Zif268 Stabilizes the Domain Structure and Mediates DNA Binding

Biochemistry ◽  
2011 ◽  
Vol 50 (28) ◽  
pp. 6266-6272 ◽  
Author(s):  
Shigeru Negi ◽  
Miki Imanishi ◽  
Maeko Sasaki ◽  
Kazuya Tatsutani ◽  
Shiroh Futaki ◽  
...  
Keyword(s):  
Dna Binding ◽  
Domain Structure ◽  
Arginine Residue ◽  
Leucine Residue ◽  
Recognition Helix

scholarly journals The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix.

1994 ◽  
Vol 14 (4) ◽  
pp. 2755-2766 ◽  
Author(s):  
D G Overdier ◽  
A Porcella ◽  
R H Costa
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Hepatocyte Nuclear Factor ◽  
Binding Properties ◽  
Winged Helix ◽  
Amino Acid Region ◽  
Dna Binding Specificity ◽  
Recognition Helix ◽  
Dna Binding Properties ◽  
Acid Region

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3 alpha, -3 beta, and -3 gamma) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3 beta protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3 beta protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3 beta with corresponding residues from HFH-1 enabled the HNF-3 beta recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.

Effects of Bulkiness and Hydrophobicity of an Aliphatic Amino Acid in the Recognition Helix of the GAGA Zinc Finger on the Stability of the Hydrophobic Core and DNA Binding Affinity†

Biochemistry ◽  
2008 ◽  
Vol 47 (45) ◽  
pp. 11717-11724 ◽  
Author(s):  
Muthu Dhanasekaran ◽  
Shigeru Negi ◽  
Miki Imanishi ◽  
Michiko Suzuki ◽  
Yukio Sugiura
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Binding Affinity ◽  
Zinc Finger ◽  
Hydrophobic Core ◽  
Aliphatic Amino Acid ◽  
Dna Binding Affinity ◽  
Recognition Helix ◽  
The Stability

scholarly journals The Oncogenic Potential of the Pax3-FKHR Fusion Protein Requires the Pax3 Homeodomain Recognition Helix but Not the Pax3 Paired-Box DNA Binding Domain

1999 ◽  
Vol 19 (1) ◽  
pp. 594-601 ◽  
Author(s):  
Paula Y. P. Lam ◽  
Jack E. Sublett ◽  
Andrew D. Hollenbach ◽  
Martine F. Roussel
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Chromosomal Translocation ◽  
Cellular Transformation ◽  
Small Region ◽  
Transactivation Domain ◽  
Recognition Helix

ABSTRACT The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis revealed that both the third α-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. Our results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein.

The Oncogenic Potential of the Pax3-FKHR Fusion Protein Requires the Pax3 Homeodomain Recognition Helix but Not the Pax3 Paired-Box DNA Binding Domain

1999 ◽  
Vol 19 (10) ◽  
pp. 7287-7287
Author(s):  
Paula Y.P. Lam ◽  
Jack E. Sublett ◽  
Andrew D. Hollenbach ◽  
Martine Roussel
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Oncogenic Potential ◽  
Recognition Helix

scholarly journals The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix

1994 ◽  
Vol 14 (4) ◽  
pp. 2755-2766
Author(s):  
D G Overdier ◽  
A Porcella ◽  
R H Costa
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Hepatocyte Nuclear Factor ◽  
Binding Properties ◽  
Winged Helix ◽  
Amino Acid Region ◽  
Dna Binding Specificity ◽  
Recognition Helix ◽  
Dna Binding Properties ◽  
Acid Region

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3 alpha, -3 beta, and -3 gamma) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3 beta protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3 beta protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3 beta with corresponding residues from HFH-1 enabled the HNF-3 beta recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.

Biochemical and In Vivo Characterization of Amino Acid Substitutions in the Runx1 (AML1) Runt Domain Found in FPD/AML, AML M0, and Cleidocranial Dysplasia (CCD) Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 464-464
Author(s):  
Christina J. Matheny ◽  
Takeshi Corpora ◽  
Maren E. Speck ◽  
Ting-Lei Gu ◽  
John H. Bushweller ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Domain Structure ◽  
Cleidocranial Dysplasia ◽  
Amino Acid Substitutions ◽  
Runt Domain ◽  
Core Binding Factor ◽  
Binding Factor

Abstract Runx1 and CBF β are the DNA-binding and non DNA-binding subunits of a core-binding factor that is required for hematopoiesis, and that is frequently mutated in leukemia. Runx2 is the DNA-binding subunit of a core-binding factor required for bone formation. Mono-allelic deletion, nonsense, frameshift, and missense mutations have been found in RUNX1 in familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML) and in myelodysplastic syndrome (MDS), and biallelic mutations in RUNX1 are found in 20% of AML M0 patients. Similar types of mono-allelic mutations have been found in RUNX2 in patients with cleidocranial dysplasia (CCD), an inherited skeletal syndrome. FPD/AML and CCD pedigrees have revealed varying degrees of disease severity depending on the nature of the specific mutation. Additionally, it has been observed that mutations involving amino acids in the DNA binding Runt domain that directly contact DNA are associated primarily with Runx1 and hematopoietic disorders, while mutations predicted to disrupt CBF β binding or the Runt domain structure are found only in Runx2 in CCD patients. We introduced 21 amino acid substitutions into the Runt domain of Runx1 identified in FPD/AML, AML M0, and CCD patients, and quantified their effects on DNA binding, heterodimerization with CBFβ, and the Runt domain structure using yeast one- and two-hybrid, quantitative electrophoretic mobility shift, heteronuclear single quantum correlation spectroscopy, and urea denaturation experiments. To address the impact on in vivo function, several of these point mutations were engineered into the endogenous Runx1 allele in mice. These five mutations include: R177X, R174Q, T149A, T161A, and L148F. R177X is found in FPD/AML patients and truncates Runx1 two amino acids before the C-terminal boundary of the Runt domain. R174Q (found in FPD/AML and CCD) disrupts DNA binding 1000-fold, but does not disrupt CBFb binding or perturb the Runt domain fold. T149A (found only in CCD) disrupts CBFβ binding 13-fold while T161A (not found in patients) disrupts CBFβ binding 40-fold. Both T149A and T161A slightly perturb the Runt domain fold, but do not alter DNA binding affinity. L148F (found in CCD) also disrupts the Runt domain fold, and decreases DNA binding. All animals heterozygous for these alleles are viable. Mice homozygous for R177X and R174Q die during gestation. Mice homozygous for the T149A and T161A mutations, on the other hand, are born at normal Mendelian frequencies, but 62% and 100%, respectively, die by or at three weeks of age from an undetermined cause. The effects of these mutations on hematopoietic progenitor and platelet numbers, both of which are affected in FPD/AML patients, will be presented. We conclude that mutations that affect CBFβ binding result in hypomorphic Runx1 alleles, while mutations involving DNA contacts result in more severe inactivation of Runx1 function. Thus FPD/AML, AML M0, and MDS require mutations that severely inactivate Runx1 function, while CCD can result from more subtle alterations in Runx2.

scholarly journals Phage P4 origin-binding domain structure reveals a mechanism for regulation of DNA-binding activity by homo- and heterodimerization of winged helix proteins

2002 ◽  
Vol 43 (4) ◽  
pp. 855-867 ◽  
Author(s):  
Hye-Jeong Yeo ◽  
Gunter Ziegelin ◽  
Sergey Korolev ◽  
Richard Calendar ◽  
Erich Lanka ◽  
...  
Keyword(s):  
Dna Binding ◽  
Domain Structure ◽  
Binding Activity ◽  
Binding Domain ◽  
Winged Helix ◽  
Dna Binding Activity
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