Inhibition of transcription factor assembly and structural stability on mitoxantrone binding with DNA

2010 ◽  
Vol 30 (5) ◽  
pp. 331-340 ◽  
Author(s):  
Shahper N. Khan ◽  
Mohd Danishuddin ◽  
Asad U. Khan
Keyword(s):  
Transcription Factor ◽  
Binding Mode ◽  
Cd Spectroscopy ◽  
Sequence Specificity ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Naked Dna ◽  
Plasmid Nicking Assay ◽  
Modelling Studies ◽  
Mobility Shift Assay

MTX (mitoxantrone) is perhaps the most promising drug used in the treatment of various malignancies. Comprehensive literature on the therapeutics has indicated it to be the least toxic in its class, although its mechanism of action is still not well defined. In the present study, we have evaluated the associated binding interactions of MTX with naked DNA. The mechanism of MTX binding with DNA was elucidated by steady-state fluorescence and a static-type quenching mechanism is suggested for this interaction. Thermodynamic parameters from van 't Hoff plots showed that the interaction of these drugs with DNA is an entropically driven phenomenon. The binding mode was expounded by attenuance measurements and competitive binding of a known intercalator. Sequence specificity of these drug–DNA complexes was analysed by FTIR (Fourier-transform infrared) spectroscopy and molecular modelling studies. CD spectroscopy and the plasmid nicking assay showed that the binding of this drug with DNA results in structural and conformational perturbations. EMSA (electrophoretic mobility-shift assay) results showed that these drug–DNA complexes prevent the binding of octamer TF (transcription factor) to DNA. In summary, the study implicates MTX-induced conformational instability and transcription inhibition on DNA binding.

scholarly journals Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf

1995 ◽  
Vol 311 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M A Bevilacqua ◽  
M C Faniello ◽  
P D′Agostino ◽  
B Quaresima ◽  
M T Tiano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mobility Shift ◽  
Differentiated Cells ◽  
Binding Factor ◽  
H Gene ◽  
Ferritin Gene ◽  
Mobility Shift Assay ◽  
Promoter Interaction

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.

scholarly journals PgMYB2, a MeJA-Responsive Transcription Factor, Positively Regulates the Dammarenediol Synthase Gene Expression in Panax Ginseng

2019 ◽  
Vol 20 (9) ◽  
pp. 2219 ◽  
Author(s):  
Tuo Liu ◽  
Tiao Luo ◽  
Xiangqian Guo ◽  
Xian Zou ◽  
Donghua Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Panax Ginseng ◽  
Growth Regulation ◽  
Ginseng Root ◽  
Myb Transcription Factor ◽  
Mobility Shift ◽  
Binding Interaction ◽  
Mobility Shift Assay ◽  
Dammarenediol Synthase

The MYB transcription factor family members have been reported to play different roles in plant growth regulation, defense response, and secondary metabolism. However, MYB gene expression has not been reported in Panax ginseng. In this study, we isolated a gene from ginseng adventitious root, PgMYB2, which encodes an R2R3-MYB protein. Subcellular localization revealed that PgMYB2 protein was exclusively detected in the nucleus of Allium cepa epidermis. The highest expression level of PgMYB2 was found in ginseng root and it was significantly induced by plant hormones methyl jasmonate (MeJA). Furthermore, the binding interaction between PgMYB2 protein and the promoter of dammarenediol synthase (DDS) was found in the yeast strain Y1H Gold. Moreover, the electrophoretic mobility shift assay (EMSA) identified the binding site of the interaction and the results of transiently overexpressing PgMYB2 in plants also illustrated that it may positively regulate the expression of PgDDS. Based on the key role of PgDDS gene in ginsenoside synthesis, it is reasonable to believe that this report will be helpful for the future studies on the MYB family in P. ginseng and ultimately improving the ginsenoside production through genetic and metabolic engineering.

scholarly journals Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ.

1992 ◽  
Vol 12 (1) ◽  
pp. 413-421 ◽  
Author(s):  
P Cortes ◽  
O Flores ◽  
D Reinberg
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Stable Complex ◽  
Mobility Shift ◽  
Yeast Saccharomyces Cerevisiae ◽  
Gel Mobility Shift Assay ◽  
Two Factors ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Tfiid Complex

The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex. The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP. Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect. TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP. TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa. These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence.

scholarly journals Initiation of Base Excision Repair of Oxidative Lesions in Nucleosomes by the Human, Bifunctional DNA Glycosylase NTH1

2007 ◽  
Vol 27 (24) ◽  
pp. 8442-8453 ◽  
Author(s):  
Amalthiya Prasad ◽  
Susan S. Wallace ◽  
David S. Pederson
Keyword(s):  
Base Excision Repair ◽  
Excision Repair ◽  
Minor Groove ◽  
Dna Glycosylase ◽  
Mobility Shift ◽  
Naked Dna ◽  
Histone Octamer ◽  
Mobility Shift Assay ◽  
Base Excision ◽  
Gel Mobility Shift

ABSTRACT Oxidative lesions account for much of the spontaneously occurring DNA damage in normal cells and, left unrepaired, can be mutagenic or cytotoxic. We have investigated the capacity of purified human enzymes to initiate the base excision repair (BER) of oxidative lesions in model nucleosomes. In a construct where the minor groove of a thymine glycol lesion faced outward from the histone octamer, the human DNA glycosylase NTH1 (hNTH1) processed the lesion with nearly the same efficiency as in naked DNA. The hNTH1 reaction did not generate free DNA, indicating that the first step in BER occurred without irreversibly disrupting nucleosomes. Instead, lesion processing entailed the formation of nucleosome-hNTH1 ternary complexes that could be visualized in a gel mobility shift assay. These complexes contained both processed and unprocessed DNA. hNTH1 processing of lesions whose minor groove faced toward the histone octamer was poor at low hNTH1 concentrations but increased substantially as hNTH1 concentrations increased to nearly physiological levels. Additionally, an inward-facing lesion near the nucleosome edge was more efficiently processed than one closer to the nucleosome dyad. These observations suggest that access to sterically occluded lesions entails the partial, reversible unwrapping of DNA from the histone octamer, allowing hNTH1 to capture its DNA substrate when it is in an unwound state.

scholarly journals The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase

2020 ◽  
pp. jbc.RA120.015824
Author(s):  
Manisha Yadav ◽  
Ravi Shankar Singh ◽  
Daniel Hogan ◽  
Venkatasubramanian Vidhyasagar ◽  
Shizhuo Yang ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Mutational Analysis ◽  
Sequence Specificity ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Dead Box ◽  
Kh Domain ◽  
Mobility Shift Assay ◽  
Exponential Enrichment ◽  
Binding Sequence

The K-homology (KH) domain is a nucleic acid binding domain present in many proteins. Recently we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding single-stranded (ss)DNA and ssRNA. Electrophoretic mobility shift assay (EMSA) and nuclear magnetic resonance (NMR) revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG-loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, and crosslinking immunoprecipitation (CLIP)-seq showed that the KH domain binds C/T rich DNA and U rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-HSQC NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single strand tails, and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.

Transcription factor binding study by capillary zone electrophoretic mobility shift assay

2003 ◽  
Vol 24 (12) ◽  
pp. 96-100 ◽  
Author(s):  
Zsolt Ronai ◽  
Yan Wang ◽  
Julia Khandurina ◽  
Paul Budworth ◽  
Maria Sasvari-Szekely ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Transcription Factor Binding ◽  
Binding Study ◽  
Mobility Shift ◽  
Factor Binding ◽  
Capillary Zone ◽  
Mobility Shift Assay

scholarly journals Novel DNA Bis-intercalation by MLN944, a Potent Clinical Bisphenazine Anticancer Drug

2004 ◽  
Vol 279 (44) ◽  
pp. 46096-46103 ◽  
Author(s):  
Jixun Dai ◽  
Chandanamalie Punchihewa ◽  
Prakash Mistry ◽  
Aik Teong Ooi ◽  
Danzhou Yang
Keyword(s):  
Dna Binding ◽  
Anticancer Drug ◽  
Topoisomerase I ◽  
Specific Binding ◽  
Binding Mode ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Phase I Clinical Trials ◽  
Mobility Shift ◽  
Dna Sequence Specificity

The new bisphenazine anticancer drug MLN944 is a novel cytotoxic agent with exceptional anti-tumor activity against a range of human and murine tumor models both invitroand invivo. MLN944 has recently entered Phase I clinical trials. Despite the structural similarity with its parent monophenazine carboxamide and acridine carboxamide anticancer compounds, MLN944 appears to work by a distinct mechanism of inhibiting DNA transcription rather than the expected mechanism of topoisomerase I and II inhibition. Here we present the first NMR structure of MLN944 complexed with d(ATGCAT)2DNA duplex, demonstrating a novel binding mode in which the two phenazine rings bis-intercalate at the 5′-TpG site, with the carboxamide amino linker lying in the major groove of DNA. The MLN944 molecule adopts a significantly unexpected conformation and side chain orientation in the DNA complex, with the N10 on the phenazine ring protonated at pH 7. The phenazine chromophore of MLN944 is very well stacked with the flanking DNA base pairs using the parallel base-stacking intercalation binding mode. The DNA sequence specificity and the groove recognition of MLN944 binding is determined by several site-specific hydrogen bond interactions with the central G:C base pair as well as the favorable stacking interactions with the 5′-flanking thymine. The specific binding site of MLN944 is known to be recognized by a number of important transcription factors. Our electrophoretic gel mobility shift assay results demonstrated that the c-Jun DNA binding to the AP-1 site is significantly inhibited by MLN944 in a dose-dependent manner. Thus, the exceptional biological activity of MLN944 may be due to its novel DNA binding mode leading to a unique mechanism of action.

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