scholarly journals Structures of 1:1 and 2:1 complexes of BMVC and MYC promoter G-quadruplex reveal a mechanism of ligand conformation adjustment for G4-recognition

2019 ◽  
Author(s):  
Wenting Liu ◽  
Clement Lin ◽  
Guanhui Wu ◽  
Jixun Dai ◽  
Ta-Chau Chang ◽  
...  
Keyword(s):  
Exchange Rates ◽  
Specific Binding ◽  
Sequence Specificity ◽  
Base Plane ◽  
Solution Structures ◽  
Myc Oncogene ◽  
High Affinity ◽  
G Quadruplex ◽  
Ligand Conformation

AbstractBMVC is the first fluorescent probe designed to detect G-quadruplexes (G4s) in vivo. The MYC oncogene promoter forms a G4 (MycG4) which acts as a transcription silencer. Here, we report the high-affinity and specific binding of BMVC to MycG4 with unusual slow-exchange rates on the NMR timescale. We also show that BMVC represses MYC in cancer cells. We determined the solution structures of the 1:1 and 2:1 BMVC–MycG4 complexes. BMVC first binds the 5′-end of MycG4 to form a 1:1 complex with a well-defined structure. At higher ratio, BMVC also binds the 3′-end to form a second complex. In both complexes, the crescent-shaped BMVC recruits a flanking DNA residue to form a BMVC-base plane stacking over the external G-tetrad. Remarkably, BMVC adjusts its conformation to a contracted form to match the G-tetrad for an optimal stacking interaction. This is the first structural example showing the importance of ligand conformational adjustment in G4 recognition. BMVC binds the more accessible 5′-end with higher affinity, whereas sequence specificity is present at the weaker-binding 3′-site. Our structures provide insights into specific recognition of MycG4 by BMVC and useful information for design of G4-targeted anticancer drugs and fluorescent probes.

scholarly journals AID Associates with Single-Stranded DNA with High Affinity and a Long Complex Half-Life in a Sequence-Independent Manner

2006 ◽  
Vol 27 (1) ◽  
pp. 20-30 ◽  
Author(s):  
Mani Larijani ◽  
Alexander P. Petrov ◽  
Oxana Kolenchenko ◽  
Maribel Berru ◽  
Sergey N. Krylov ◽  
...  
Keyword(s):  
Half Life ◽  
Dissociation Constants ◽  
Hot Spot ◽  
Scaffolding Protein ◽  
Sequence Specificity ◽  
Independent Manner ◽  
Single Stranded Dna ◽  
Stem Loop ◽  
High Affinity

ABSTRACT Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and single-stranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors.

scholarly journals High Affinity binding of Yeast Nucleolin Nsr1 to Co-transcriptionally Formed G4 DNA Obstructs Replication and Elevates Genome Instability

2019 ◽  
Author(s):  
Shivani Singh ◽  
Alexandra Berroyer ◽  
Minseon Kim ◽  
Nayun Kim
Keyword(s):  
Dna Binding ◽  
Genome Instability ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Yeast Genome ◽  
Stable Complex ◽  
High Affinity ◽  
G4 Dna ◽  
G Quadruplex

ABSTRACTA significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin NCL is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that G4 DNA-binding is a conserved function in the yeast nucleolin Nsr1. Furthermore, we demonstrate that the Nsr1-G4 DNA complex formation results in replication obstruction and is a major factor in inducing the genome instability associated with the co-transcriptionally formed G4 DNA in the yeast genome. The G4-associated genome instability and the G4 DNA-binding in vivo requires the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vitro and in vivo and restores unhindered replication, overall resulting in a sharp reduction in the G4-associated genome instability. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and obstructing replication.AUTHOR SUMMARYGenome instability is uniquely elevated at sequences containing multiple runs of guanines, which can fold into the unusual, four-stranded G-quadruplex (G4) DNA. In this study, we report a novel finding that a highly conserved G4 DNA binding protein Nsr1 can elevate the rate of recombination and chromosomal rearrangement occurring at a G4 DNA-forming sequence in the genome of Saccharomyces cerevisiae. The elevated genome instability requires the C-terminally located RGG domain of Nsr1, which supports the high-affinity interaction between the protein and G4 DNA. The connection between G4-specific genome instability and the function of Nsr1 to form stable complex with G4 DNA led to the hypothesis that the high-affinity Nsr1-G4 DNA complexes can become a barrier to replication. We demonstrate here that the presence of Nsr1 in fact slows the replication past a G4 DNA-containing genomic site and that the RGG domain is required to facilitate such replication block.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals Loss of Striatal [76Br]Bromospiperone Binding Sites Demonstrated by Positron Tomography in Progressive Supranuclear Palsy

1986 ◽  
Vol 6 (2) ◽  
pp. 131-136 ◽  
Author(s):  
J. C. Baron ◽  
B. Mazière ◽  
C. Loc'h ◽  
H. Cambon ◽  
P. Sgouropoulos ◽  
...  
Keyword(s):  
Progressive Supranuclear Palsy ◽  
Binding Sites ◽  
Matched Control ◽  
Specific Binding ◽  
Neuroleptic Drug ◽  
Uptake Ratio ◽  
Positron Tomography ◽  
High Affinity ◽  
Da Receptors

Using positron tomography and 76Br-labeled bromospiperone, a neuroleptic drug with high affinity for the dopamine (DA) receptors, we have estimated the specific binding of the radiotracer to striatal DA receptors in seven patients suffering from progressive supranuclear palsy. Compared with age- and sex-matched control subjects, we found a significant (p < 0.02) decrease of the striatum–cerebellum uptake ratio in progressive supranuclear palsy patients, suggesting loss of striatal DA receptors. This in vivo study confirms recent postmortem data on progressive supranuclear palsy patients and provides an explanation for the lack of benefit from l-DOPA and DA agonists in this condition, despite reduced nigrostriatal dopaminergic function.

scholarly journals Engineering yellow fluorescent protein probe for visualization of parallel DNA G-quadruplex

2018 ◽  
Vol 21 (3) ◽  
pp. 84-89 ◽  
Author(s):  
Tuom TT Truong ◽  
Trang PT Phan ◽  
Linh TT Le ◽  
Dung H Nguyen ◽  
Hoang D Nguyen ◽  
...  
Keyword(s):  
Small Molecules ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Biological Processes ◽  
Naked Eye ◽  
High Affinity ◽  
Quadruplex Structure ◽  
G Quadruplex

Introduction: The formation of G-quadruplex plays a key role in many biological processes. Therefore, visualization of G-quadruplex is highly essential for design of G-quadruplex-targeted small molecules (drugs). Herein, we report on an engineered fluorescent protein probe which was able to distinguish G-quadruplex topologies. Methods: The fluorescent protein probe was generated by genetically incorporating yellow fluorescent protein (YFP) to RNA helicase associated with AU-rich element (RHAU) peptide motif. Results: This probe could selectively bind and visualize parallel G-quadruplex structure (T95-2T) at high affinity (Kd~130 nM). Visualization of the parallel G-quadruplex by RHAU-YFP could be easily observed in vitro by using normal Gel Doc or the naked eye. Conclusion: The YFP probe could be encoded in cells to provide a powerful tool for detection of parallel G-quadruplexes both in vitro and in vivo.  

scholarly journals A high-affinity oestrogen-binding protein in cockerel liver cytosol

1979 ◽  
Vol 180 (2) ◽  
pp. 347-353 ◽  
Author(s):  
C B Lazier ◽  
A J Haggarty
Keyword(s):  
Binding Sites ◽  
Proteinase Inhibitors ◽  
Specific Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Binding Activity ◽  
Single Class ◽  
High Affinity ◽  
Significant Concentration

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1–4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

scholarly journals Contribution of gangliosides to thyroxin binding with plasma membranes

1995 ◽  
Vol 41 (2) ◽  
pp. 28-30
Author(s):  
T. S. Saatov ◽  
F. Ya. Gulyamova ◽  
G. U. Usmanova
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Specific Binding ◽  
Brain Cell ◽  
Cell Recognition ◽  
High Affinity ◽  
Specific Binding Sites

Besides intracellular receptors of thyroid hormones, specific binding sites for T3 and T4 were detected on plasma membranes (PM) of some cells and a relationship between membrane reception .and lipid composition of membranes shown. The parameters of 125I-T4 binding to highly purified PM of hepatic and cerebral cells of rats were studied. The hepatic and cerebral cellular membranes were found to contain two sites of hormone binding each, one of these sites being characterized by a high affinity and low capacity, and the other by low affinity and a higher binding capacity. The association constant of highly affine site of hepatocyte membranes was found to be higher than that of brain cell membranes. T4 membranous receptors may be significant in the process of cell “recognition" by the hormone. In vivo and in vitro experiments with 125I-T4 and 14C-labeled thyroxin in ganglioside fractions showed appreciable binding of the hormone to Gm3 fraction, this evidently pointing to participation of this, ganglioside in T4 interaction with membrane receptor. It is possible that gangliosides situated on membranous surface are components of or function as receptors.

scholarly journals Specific Binding of Anionic Porphyrin and Phthalocyanine to the G-Quadruplex with a Variety of in Vitro and in Vivo Applications

Molecules ◽  
2012 ◽  
Vol 17 (9) ◽  
pp. 10586-10613 ◽  
Author(s):  
Hidenobu Yaku ◽  
Takashi Murashima ◽  
Daisuke Miyoshi ◽  
Naoki Sugimoto
Keyword(s):  
Specific Binding ◽  
G Quadruplex

scholarly journals Rap1 in Candida albicans: an Unusual Structural Organization and a Critical Function in Suppressing Telomere Recombination

2009 ◽  
Vol 30 (5) ◽  
pp. 1254-1268 ◽  
Author(s):  
Eun Young Yu ◽  
Wei-Feng Yen ◽  
Olga Steinberg-Neifach ◽  
Neal F. Lue
Keyword(s):  
Candida Albicans ◽  
Telomere Length ◽  
Specific Binding ◽  
Repeat Unit ◽  
Gene Promoters ◽  
Sequence Specificity ◽  
Null Mutant ◽  
Ribosomal Protein Genes

ABSTRACT Rap1 (repressor activator protein 1) is a conserved multifunctional protein initially identified as a transcriptional regulator of ribosomal protein genes in Saccharomyces cerevisiae but subsequently shown to play diverse functions at multiple chromosomal loci, including telomeres. The function of Rap1 appears to be evolutionarily plastic, especially in the budding yeast lineages. We report here our biochemical and molecular genetic characterizations of Candida albicans Rap1, which exhibits an unusual, miniaturized domain organization in comparison to the S. cerevisiae homologue. We show that in contrast to S. cerevisiae, C. albicans RAP1 is not essential for cell viability but is critical for maintaining normal telomere length and structure. The rap1 null mutant exhibits drastic telomere-length dysregulation and accumulates high levels of telomere circles, which can be largely attributed to aberrant recombination activities at telomeres. Analysis of combination mutants indicates that Rap1 and other telomere proteins mediate overlapping but nonredundant roles in telomere protection. Consistent with the telomere phenotypes of the mutant, C. albicans Rap1 is localized to telomeres in vivo and recognizes the unusual telomere repeat unit with high affinity and sequence specificity in vitro. The DNA-binding Myb domain of C. albicans Rap1 is sufficient to suppress most of the telomere aberrations observed in the null mutant. Notably, we were unable to detect specific binding of C. albicans Rap1 to gene promoters in vivo or in vitro, suggesting that its functions are more circumscribed in this organism. Our findings provide insights on the evolution and mechanistic plasticity of a widely conserved and functionally critical telomere component.

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