Solution structures of the trihelix DNA-binding domains of the wild-type and a phosphomimetic mutant of Arabidopsis GT-1: Mechanism for an increase in DNA-binding affinity through phosphorylation

2010 ◽  
Vol 78 (14) ◽  
pp. 3033-3047 ◽  
Author(s):  
Takashi Nagata ◽  
Emi Niyada ◽  
Natsuki Fujimoto ◽  
Yuuya Nagasaki ◽  
Kazuaki Noto ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Wild Type ◽  
Solution Structures ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Binding Affinity

scholarly journals The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of Staphylococcus aureus

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Ivan Birukou ◽  
Nam K. Tonthat ◽  
Susan M. Seo ◽  
Bryan D. Schindler ◽  
Glenn W. Kaatz ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Family Member ◽  
Binding Affinity ◽  
Multidrug Resistant ◽  
Content Type ◽  
Linker Region ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Binding Affinity

ABSTRACTOverexpression of theStaphylococcus aureusmultidrug efflux pump MepA confers resistance to a wide variety of antimicrobials.mepAexpression is controlled by MarR family member MepR, which repressesmepAand autorepresses its own production. Mutations inmepRare a primary cause ofmepAoverexpression in clinical isolates of multidrug-resistantS. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasingmepRoperator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistantStaphylococcus aureus.IMPORTANCEStaphylococcus aureusis a major health threat to immunocompromised patients.S. aureusmultidrug-resistant variants that overexpress the multidrug efflux pumpmepAemerge frequently due to point mutations in MarR family member MepR, themepAtranscription repressor. Significantly, the majority of MepR mutations identified in theseS. aureusclinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistantStaphylococcus aureus.

scholarly journals Sequence-dependent contribution of distal binding domains to CAP protein-DNA binding affinity

1991 ◽  
Vol 19 (3) ◽  
pp. 611-616 ◽  
Author(s):  
Dennise D. Dalma-Weiszhausz ◽  
Marc R. Gartenberg ◽  
Donald M. Crothers
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Binding Domains ◽  
Sequence Dependent ◽  
Dna Binding Affinity

Spectroscopic Determination of the Binding Affinity of Zinc to the DNA-Binding Domains of Nuclear Hormone Receptors†

Biochemistry ◽  
2003 ◽  
Vol 42 (48) ◽  
pp. 14214-14224 ◽  
Author(s):  
John C. Payne ◽  
Brian W. Rous ◽  
Adam L. Tenderholt ◽  
Hilary Arnold Godwin
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Hormone Receptors ◽  
Nuclear Hormone Receptors ◽  
Spectroscopic Determination ◽  
Dna Binding Domains ◽  
Binding Domains

Targeting DNA Repair Gene, RAD52, Induces Exhaustion of the Proliferating CML-CP Leukemia Stem Cells Carrying Oxidative DNA Damage

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 447-447
Author(s):  
Kimberly Cramer ◽  
Elisabeth Bolton ◽  
Margaret Nieborowska-Skorska ◽  
Sylwia Flis ◽  
Tomasz Skorski
Keyword(s):  
Stem Cells ◽  
Dna Binding ◽  
Leukemia Stem Cells ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Dsb Repair ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Fold Reduction ◽  
Clonogenic Potential

Abstract Abstract 447 BCR-ABL1 transforms hematopoietic stem cells (HSCs) into leukemia stem cells (LSCs) to induce chronic myeloid leukemia in chronic phase (CML-CP). We detected that the most primitive LSCs display elevated levels of reactive oxygen species (ROS) and accumulate excessive numbers of potentially lethal DNA double-strand breaks (DSBs). We also reported that BCR-ABL1-transformed cells exhibit enhanced RAD51-mediated homologous recombination repair (HRR) activity occurring in S and G2/M cell cycle phases. In normal cells initiation of RAD51-mediated HRR is directed either by BRCA1– or RAD52–dependent mechanisms. Since BCR-ABL1 kinase downregulated BRCA1, LSCs containing high number of DSBs should depend more on RAD52 to promote HRR to repair lethal DSBs. We found that in vivo leukemogenic potential of BCR-ABL1 –positive RAD52−/− hematopoietic cells is abrogated in comparison to their BCR-ABL1 -positive RAD52+/+ counterparts. The absence of RAD52 in BCR-ABL1 –positive cells reduced the percentage of Lin−Kit+Sca1+ cells by >2-fold and inhibited their clonogenic potential and proliferation by >10-fold. In addition RAD52 knockout caused approximately 2-fold reduction of Lin−Kit+Sca1+CD34−Flt3− long-term LSCs (LT-LSCs) and Lin−Kit+Sca1+CD34+Flt3− short-term LSCs (ST-LSCs). Conversely, 4-fold accumulation of BCR-ABL1 –positive RAD52−/− Lin−Kit+Sca1+eFluor670max quiescent cells was detected in comparison to BCR-ABL1 –positive RAD52+/+ counterparts. These effects were accompanied by 2-fold reduction of the percentage of BCR-ABL1 –positive RAD52−/− cells in S and G2/M and 7-fold increase of these cells in sub-G1 when compared to BCR-ABL1 –positive RAD52+/+ counterparts. BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells accumulated more DSBs than BCR-ABL1 –positive RAD52+/+ cells. These differences were not observed between non-transformed RAD52−/− and RAD52+/+ cells. Expression of the wild-type RAD52 reduced the accumulation of lethal DSBs and rescued the clonogenic potential and proliferation of BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells. Downregulation of ROS with antioxidants vitamin E (VE) and N-acetyl-cysteine (NAC) exerted similar effect as restored expression of RAD52. Thus it appears that RAD52 is necessary to repair the extensive ROS-induced DSBs in LSC-enriched Lin−Kit+Sca1+ cells. BCR-ABL1 kinase does not affect the expression of RAD52 protein, but phosphorylates RAD52 on Y104. However, expression of RAD52(Y104F) phosphorylation-less mutant reduced the number of DSBs and rescued the clonogenic potential of BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells in a similar way to the wild-type RAD52. Accordingly, RAD52-mediated DSB repair activity in CML-CP cells should not be affected by imatinib treatment. RAD52 mediates the annealing of complementarry DNA strands during DSB repair. To exert this function RAD52 has two DNA binding domains. Expression of RAD52(F79A) and RAD52(K102A) DNA binding-deficient mutants (each amino acid substitution inactivated different DNA binding domain) failed to prevent the accumulation of DSBs and did not rescue the clonogenic and proliferative potential of BCR-ABL1-positive RAD52−/− cells. In addition, RAD52(F79A), but not RAD52(Y104F) inhibited DSB repair by HRR. Therefore DNA binding capability of RAD52 appears essential for BCR-ABL1 –mediated leukemogenesis, but it is dispensable in normal hematopoietic cells. The “addiction” of BCR-ABL1 leukemia cells to RAD52 was confirmed by demonstration that RAD52(F79A) mutant inhibited clonogenic potential of CD34+ CML-CP cells, but not normal counterparts. Furthermore, to determine if RAD52 DNA binding domains could be targeted to selectively inhibit CML-CP, peptide aptamers containing RAD52 DNA binding domain amino acids sequence surrounding F79 were employed as potential decoys for RAD52 DNA binding. Aptamer containing F79, but not the A79 substitution, diminished the number of RAD52 foci and reduced the clonogenic potential and proliferation of CD34+ cells from CML-CP, but not from normal donors. In conclusion, we postulate that RAD52 is essential for BCR-ABL1 –mediated leukemogenesis and that DNA binding domains of RAD52 may be targeted for selective elimination of the proliferating CML-CP LSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.

1995 ◽  
Vol 15 (3) ◽  
pp. 1522-1535 ◽  
Author(s):  
W J Fredericks ◽  
N Galili ◽  
S Mukhopadhyay ◽  
G Rovera ◽  
J Bennicelli ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Fusion Protein ◽  
Target Genes ◽  
Transcriptional Activator ◽  
Wild Type ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Pediatric Solid Tumors ◽  
Single Polypeptide

Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes.

Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein

2009 ◽  
Vol 39 (3) ◽  
pp. 195-201 ◽  
Author(s):  
Hee Keun Lee ◽  
Mee Hyun Song ◽  
Myengmo Kang ◽  
Jung Tae Lee ◽  
Kyoung-Ah Kong ◽  
...  
Keyword(s):  
Dna Binding ◽  
Inner Ear ◽  
Molecular Mechanisms ◽  
Tertiary Structure ◽  
In Vitro Assays ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dna Binding Domains ◽  
Binding Domains

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

A Novel Zinc-binding Motif Revealed by Solution Structures of DNA-binding Domains of Arabidopsis SBP-family Transcription Factors

2004 ◽  
Vol 337 (1) ◽  
pp. 49-63 ◽  
Author(s):  
Kazuhiko Yamasaki ◽  
Takanori Kigawa ◽  
Makoto Inoue ◽  
Masaru Tateno ◽  
Tomoko Yamasaki ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Zinc Binding ◽  
Binding Motif ◽  
Solution Structures ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Zinc Binding Motif

Quantitative Analysis of DNA Binding Affinity and Dimerization Properties of Wild-Type and Mutant Thyroid Hormone Receptor β1

Thyroid ◽  
2000 ◽  
Vol 10 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Teiji Takeda ◽  
Takeshi Nagasawa ◽  
Takahide Miyamoto ◽  
Kesami Minemura ◽  
Kiyoshi Hashizume ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Thyroid Hormone ◽  
Dna Binding ◽  
Binding Affinity ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Wild Type ◽  
Dna Binding Affinity

DNMT1 Cxxc Domain Can Functionally Substitute In An MLL Fusion Protein

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4193-4193
Author(s):  
Laurie E. Risner ◽  
Aravinda Kuntimaddi ◽  
John H. Bushweller ◽  
Nancy J. Zeleznik-Le
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Fusion Proteins ◽  
Binding Domain ◽  
Cpg Dna ◽  
Domain Swap ◽  
Binding Domains ◽  
Dna Binding Affinity

Abstract Abstract 4193 The MLL gene encodes a multi-domain protein that is involved in the maintenance of Hox gene expression during development and hematopoiesis, and was first identified through its involvement in chromosome translocations that cause leukemia. The CXXC domain of MLL, which is retained in leukemic MLL fusion proteins, is a cysteine rich DNA binding domain, with specificity for binding nonmethylated CpG-containing DNA, and is essential for MLL fusion proteins' oncogenic properties. We performed domain swap experiments in which CXXC domains from other proteins were swapped in to replace MLL's CXXC domain in the context of an oncogenic MLL fusion. CXXC domains from DNA methyltransferase 1 (DNMT1), CpG binding protein (CGBP), and methyl-CpG binding domain protein 1 (MBD1), as well as a methyl binding domain (MBD) from MBD1 were swapped into the MLL-AF9 fusion. These particular domains were chosen because their described CpG DNA binding capacity is either similar or different from that described for MLL. In vitro colony assays on isolated murine bone marrow progenitor cells infected with domain swapped or wild type MLL-AF9 fusion genes were performed in order to determine whether CpG binding domains from other proteins would affect the ability of MLL-AF9 to give an enhanced proliferative capacity to bone marrow progenitor cells. In vivo murine studies determined whether the different CpG binding domains alter the ability of MLL fusion proteins to cause leukemia. We predicted that the different CpG binding domains would change the strength or specificity of MLL binding to DNA, which would affect the ability of MLL-AF9 to cause leukemia. The results of both in vitro replating assays and in vivo leukemogenesis experiments have shown significant differences between the ability of various CpG DNA binding domains to function in the context of an MLL-AF9 fusion protein. MLL-AF9 containing the DNMT1 CXXC domain shows robust in vitro colony forming activity and in vivo leukemogenesis activity, similar to the oncogenic MLL-AF9 fusion. However, MLL-AF9 containing either the CXXC domain from CGBP or MBD1, or the MBD domain of MBD1 all show reduced colony forming ability and leukemogenicity in vivo. In vitro DNA binding experiments are currently being performed to directly measure and compare the DNA binding affinity of the CXXC domain from MLL to the other domain swap proteins. Preliminary data suggests that MLL CXXC has a stronger DNA binding affinity to non-methylated DNA compared to the other CXXC domains. Furthermore, the DNMT and CGBP CXXC domains both show lower affinity DNA binding compared to MLL CXXC, but they have different effects in MLL-AF9. This suggests that CXXC domain properties in addition to DNA binding affinity, perhaps including protein recruitment, also contribute to an MLL fusion protein's leukemogenic properties. Disclosures: No relevant conflicts of interest to declare.

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