The role of bridging ligands in determining DNA-binding ability and cross-linking patterns of dinuclear platinum(ii) antitumour complexes

2009 ◽  
pp. 10889 ◽  
Author(s):  
Jianhui Zhu ◽  
Miaoxin Lin ◽  
Damin Fan ◽  
Ziyi Wu ◽  
Yuncong Chen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cross Linking ◽  
Binding Ability ◽  
Bridging Ligands ◽  
Dinuclear Platinum

scholarly journals Role of ATP-binding motifs on DNA-binding activity and biological function of Rhp51, a Rad51 homologue in fission yeast

2002 ◽  
Vol 364 (3) ◽  
pp. 869-874 ◽  
Author(s):  
Woo J. KIM ◽  
Hyojin LEE ◽  
Eon J. PARK ◽  
Seung H. HONG ◽  
Sang D. PARK
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Atp Binding ◽  
Single Mutation ◽  
Binding Ability ◽  
Binding Motifs ◽  
Dna Binding Activity

Rhp51, a RecA and Rad51 homologue of Schizosaccharomyces pombe, plays a pivotal role in homologous recombination and recombinational repair. It has a set of the well-conserved type A and type B ATP-binding motifs, which are highly conserved in all RecA homologues. In a previous study [Kim, Lee, Park, Park and Park (2001) Nucleic Acids Res. 29, 1724–1732], we reported that a single mutation of the conserved lysine in A motif [Lys155→Ala (K155A)] destroyed the DNA repair ability of Rhp51 and that overexpression of this mutant protein conferred dominant negativity. In the present paper, we investigated DNA-binding properties of recombinant Rhp51 and its mutant proteins. Purified Rhp51 protein showed ATP-dependent double- and single-strand DNA-binding activities. To characterize the role of ATP-binding motifs, we generated Rhp51 K155A and Rhp51 Asp244→Gln (D244Q), which have a single amino acid substitution in A and B motifs respectively. Interestingly, K155A and D244Q mutations impaired ATP-dependent DNA binding in a different manner. K155A lost the DNA binding itself, whereas D244Q maintained the binding ability but lost the ATP dependency. However, despite the difference in DNA-binding ability, both mutations failed to rescue the methylmethane sulphonate and UV sensitivity of the rhp51Δ mutant. Together, these results suggested that not only the DNA binding but also the ATP dependence in DNA binding is required for proper in vivo functioning of Rhp51.

scholarly journals Circ-GALNT16 restrains colorectal cancer progression by enhancing the SUMOylation of hnRNPK

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Chaofan Peng ◽  
Yuqian Tan ◽  
Peng Yang ◽  
Kangpeng Jin ◽  
Chuan Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Binding ◽  
Rna Sequencing ◽  
Cancer Progression ◽  
Transcriptional Level ◽  
Binding Ability ◽  
Transcriptional Complex

Abstract Background Recent studies have investigated the role of circular RNAs (circRNAs) as significant regulatory factors in multiple cancer progression. Nevertheless, the biological functions of circRNAs and the underlying mechanisms by which they regulate colorectal cancer (CRC) progression remain unclear. Methods A novel circRNA (circ-GALNT16) was identified by microarray and qRT-PCR. A series of in vitro and in vivo phenotype experiments were performed to investigate the role of circ-GALNT16 in CRC. The FISH, RNA pulldown assay, RIP assay, RNA sequencing, coimmunoprecipitation, and ChIP were performed to investigate the molecular mechanisms of circ-GALNT16 in CRC progression. Results Circ-GALNT16 was downregulated in CRC and was negatively correlated with poor prognosis. Circ-GALNT16 suppressed the proliferation and metastatic ability of CRC cells in vitro and in vivo. Mechanistically, circ-GALNT16 could bind to the KH3 domain of heterogeneous nuclear ribonucleoprotein K (hnRNPK), which promoted the SUMOylation of hnRNPK. Additionally, circ-GALNT16 could enhance the formation of the hnRNPK-p53 complex by facilitating the SUMOylation of hnRNPK. RNA sequencing assay identified serpin family E member 1 as the target gene of circ-GALNT16 at the transcriptional level. Rescue assays revealed that circ-GALNT16 regulated the expression of Serpine1 by inhibiting the deSUMOylation of hnRNPK mediated by SUMO-specific peptidase 2 and then regulating the sequence-specific DNA binding ability of the hnRNPK-p53 transcriptional complex. Conclusions Circ-GALNT16 suppressed CRC progression by inhibiting Serpine1 expression through regulating the sequence-specific DNA binding ability of the SENP2-mediated hnRNPK-p53 transcriptional complex and might function as a biomarker and therapeutic target for CRC.

Platelet Accumulation on Fibrin-coated Polyethylene: Role of Platelet Activation and Factor XIII

1995 ◽  
Vol 73 (05) ◽  
pp. 850-856 ◽  
Author(s):  
F D Rubens ◽  
D W Perry ◽  
M W C Hatton ◽  
P D Bishop ◽  
M A Packham ◽  
...  
Keyword(s):  
Factor Xiii ◽  
Cross Linking ◽  
Internal Diameter ◽  
Static System ◽  
Graft Occlusion ◽  
Platelet Binding ◽  
Coated Surface ◽  
Platelet Accumulation ◽  
Polyethylene Tubing

SummaryPlatelet accumulation on small- and medium-calibre vascular grafts plays a significant role in graft occlusion. We examined platelet accumulation on the surface of fibrin-coated polyethylene tubing (internal diameter 0.17 cm) during 10 min of flow (l0ml/min) at high wall shear rate (764 s-1). Washed platelets labelled with 51Cr were resuspended in Tyrode solution containing albumin, apyrase and red blood cells (hematocrit 40%). When the thrombin that was used to form the fibrin-coated surface was inactivated with FPRCH2C1 before perfusion of the tubes with the platelet:red blood cell suspension, the accumulation of platelets was 59,840 ± 27,960 platelets per mm2, whereas accumulation on fibrin with residual active thrombin was 316,750 ± 32,560 platelets per mm2 (n = 4). When the fibrin on the surface was cross-linked by including recombinant factor XIII (rFXIII) in the fibrinogen solution used to prepare the fibrin-coated surface, platelet accumulation, after thrombin neutralization, was reduced by the cross-linking from 46,974 ± 9702 to 36,818 ± 7964 platelets per mm2 (n = 12, p <0.01). Platelet accumulation on tubes coated with D-dimer was ten times less than on tubes coated with D-domain; this finding also supports the observation that cross-linking of fibrin with the formation of γ-γ dimers reduces platelet accumulation on the fibrin-coated surface. Thrombin-activated platelets themselves were shown to cross-link fibrin when they had adhered to it during perfusion, or in a static system in which thrombin was used to form clots from FXIII-free fibrinogen in the presence of platelets. Thus, cross-linking of fibrin by FXIII in plasma or from platelets probably decreases the reactivity of the fibrin-containing thrombi to platelets by altering the lysine residue at or near the platelet-binding site of each of the γ-chains of the fibrinogen which was converted into the fibrin of these thrombi.

Tridegin, a Novel Peptidic Inhibitor of Factor XIIIa from the Leech, Haementeria ghilianii, Enhances Fibrinolysis In Vitro

1997 ◽  
Vol 77 (05) ◽  
pp. 0959-0963 ◽  
Author(s):  
Lisa Seale ◽  
Sarah Finney ◽  
Roy T Sawyer ◽  
Robert B Wallis
Keyword(s):  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Fibrinolytic Enzymes ◽  
Small Polypeptide ◽  
Tissue Plasminogen ◽  
Protein Cross Linking

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure.

1994 ◽  
Vol 14 (11) ◽  
pp. 7557-7568 ◽  
Author(s):  
J Zuo ◽  
R Baler ◽  
G Dahl ◽  
R Voellmy
Keyword(s):  
Transcription Factor ◽  
Heat Stress ◽  
Heat Shock ◽  
Dna Binding ◽  
Hydrophobic Interactions ◽  
Coiled Coil ◽  
Heat Shock Transcription Factor ◽  
Single Amino Acid ◽  
Binding Ability ◽  
Heat Shock Element

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.

scholarly journals Characterization of swelling behavior of carbon nano-filler modified polydimethylsiloxane composites

2021 ◽  
pp. 009524432110061
Author(s):  
Bo Yang ◽  
Balakrishnan Nagarajan ◽  
Pierre Mertiny
Keyword(s):  
Digital Image Analysis ◽  
Swelling Behavior ◽  
Optical Measurements ◽  
Natural Phenomenon ◽  
Cross Linking ◽  
Dimensional Changes ◽  
Experimental Findings ◽  
Carbon Based

Polymers may absorb fluids from their surroundings via the natural phenomenon of swelling. Dimensional changes due to swelling can affect the function of polymer components, such as in the case of seals, microfluidic components and electromechanical sensors. An understanding of the swelling behavior of polymers and means for controlling it can improve the design of polymer components, for example, for the previously mentioned applications. Carbon-based fillers have risen in popularity to be used for the property enhancement of resulting polymer composites. The present investigation focuses on the effects of three carbon-based nano-fillers (graphene nano-platelets, carbon black, and graphene nano-scrolls) on the dimensional changes of polydimethylsiloxane composites due to swelling when immersed in certain organic solvents. For this study, a facile and expedient methodology comprised of optical measurements in conjunction with digital image analysis was developed as the primary experimental technique to quantify swelling dimensional changes of the prepared composites. Other experimental techniques assessed polymer cross-linking densities and elastic mechanical properties of the various materials. The study revealed that the addition of certain carbon-based nano-fillers increased the overall swelling of the composites. The extent of swelling further depended on the organic solvent in which the composites were immersed in. Experimental findings are contrasted with published models for swelling prediction, and the role of filler morphology on swelling behavior is discussed.

scholarly journals Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krystyna Ślaska-Kiss ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Pál Albert ◽  
Ákos Csábrádi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Binding Affinity ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Dna Binding Protein ◽  
E Coli ◽  
Dna Binding Affinity ◽  
Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

scholarly journals Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos

2021 ◽  
Author(s):  
Chuan Chen ◽  
Wenqiang Liu ◽  
Jiayin Guo ◽  
Yuanyuan Liu ◽  
Xuelian Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Rrna Synthesis ◽  
Binding Ability ◽  
Transcriptome Regulation ◽  
Early Embryos ◽  
Intrinsic Mechanism ◽  
Regulatory Rnas

AbstractN6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells (ESCs), which highly depends on the m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell (2C) transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on 2C-related retrotransposons is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.

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