scholarly journals Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro

2004 ◽  
Vol 186 (11) ◽  
pp. 3374-3383 ◽  
Author(s):  
Jamie A. Caryl ◽  
Matthew C. A. Smith ◽  
Christopher D. Thomas
Keyword(s):  
Binding Specificity ◽  
Conjugal Transfer ◽  
Plasmid Nicking ◽  
Protein Relaxation

ABSTRACT The isolation of plasmid-protein relaxation complexes from bacteria is indicative of the plasmid nicking-closing equilibrium in vivo that serves to ready the plasmids for conjugal transfer. In pC221 and pC223, the components required for in vivo site- and strand-specific nicking at oriT are MobC and MobA. In order to investigate the minimal requirements for nicking in the absence of host-encoded factors, the reactions were reconstituted in vitro. Purified MobA and MobC, in the presence of Mg2+ or Mn2+, were found to nick at oriT with a concomitant phosphorylation-resistant modification at the 5′ end of nic. The position of nic is consistent with that determined in vivo. MobA, MobC, and Mg2+ or Mn2+ therefore represent the minimal requirements for nicking activity. Cross-complementation analyses showed that the MobC proteins possess binding specificity for oriT DNA of either plasmid and are able to complement each other in the nicking reaction. Conversely, nicking by the MobA proteins is plasmid specific. This suggests the MobA proteins may encode the nicking specificity determinant.

scholarly journals The role of BH3-only protein Bim extends beyond inhibiting Bcl-2–like prosurvival proteins

2009 ◽  
Vol 186 (3) ◽  
pp. 355-362 ◽  
Author(s):  
Delphine Mérino ◽  
Maybelline Giam ◽  
Peter D. Hughes ◽  
Owen M. Siggs ◽  
Klaus Heger ◽  
...  
Keyword(s):  
Family Members ◽  
Binding Specificity ◽  
In Vitro Studies ◽  
Genetic Approach ◽  
Activation Model ◽  
Bh3 Domain ◽  
Indirect Activation

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim's proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.

Posttranslational modifications of p27kip1 determine its binding specificity to different cyclins and cyclin-dependent kinases in vivo

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3691-3698 ◽  
Author(s):  
Wenqing Zhang ◽  
Daniele Bergamaschi ◽  
Boquan Jin ◽  
Xin Lu
Keyword(s):  
Cell Line ◽  
Posttranslational Modifications ◽  
Binding Specificity ◽  
Cyclin D3 ◽  
Kinase Assay ◽  
Breast Carcinoma Cell Line ◽  
Cyclin Dependent Kinases ◽  
2D Gel

AbstractUsing 2-dimensional gel electrophoresis (2D-gel) analysis, we show here that cell-cycle entry is associated with a significant increase in p27kip1 phosphorylation in human primary B cells. A similar pattern of increase in p27kip1 phosphorylation was also seen in 2 fast-growing tumor cell lines, Burkitt lymphoma cell line BL40 and breast carcinoma cell line Cal51, where inactive p27kip1 is expressed at high levels. Detailed analysis revealed for the first time that different cyclins and cyclin-dependent kinases (cdk's) interact with distinct posttranslationally modified isoforms of p27kip1 in vivo. Cyclin E but not cyclin A selectively interacts with phosphorylated p27kip1 isoforms, while cyclin D1 and D2 favor unphosphorylated p27kip1 isoforms in vivo. Interestingly, cyclin D3 and cdk4 selectively interact with phosphorylated p27kip1 in BL40 cells. Among all D-type cyclin/cdk4 and cdk6 complexes, cyclin D3/cdk4 is most active in sequestering the inhibitory activity of p27kip1 in vitro in a cyclinE/cdk2 kinase assay. This novel feature of the binding specificity of p27kip1 to cyclins and cdk's in vivo is interpreted in the context of overexpression of cyclin D3 in the presence of high levels of p27kip1 in human B-cell lymphomas with adverse clinical outcome.

scholarly journals NAD+ Modulates p53 DNA Binding Specificity and Function

2004 ◽  
Vol 24 (22) ◽  
pp. 9958-9967 ◽  
Author(s):  
Kevin G. McLure ◽  
Masatoshi Takagi ◽  
Michael B. Kastan
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Binding Specificity ◽  
Binding Activity ◽  
Vitamin B ◽  
Cancer Therapies ◽  
Dna Binding Activity ◽  
Dna Binding Specificity

ABSTRACT DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53.

scholarly journals Chromatin Accessibility Plays a Key Role in Selective Targeting of Hox Proteins

2018 ◽  
Author(s):  
Damiano Porcelli ◽  
Bettina Fischer ◽  
Steven Russell ◽  
Robert White
Keyword(s):  
Binding Specificity ◽  
Chromatin Accessibility ◽  
Binding Selectivity ◽  
Hox Proteins ◽  
Systematic Analysis ◽  
Binding Regions ◽  
Hox Protein ◽  
Accessible Chromatin

AbstractHox protein transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. Understanding the basis of Hox function has long faced the problem that, while the different members of the Hox family show clear functional specificity in vivo, they all show very similar binding specificity in vitro. Based on in vitro studies, cofactors may increase Hox binding selectivity however a satisfactory understanding of in vivo Hox target selectivity is still lacking.We have carried out a systematic analysis of the in vivo genomic binding profiles of all eight Drosophila Hox proteins using transient transfection in Kc167 cells to examine Hox protein targeting. We find that Hox proteins show considerable binding selectivity in vivo in the absence of the canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility; binding sites in less accessible chromatin show the highest selectivity and the different Hox proteins exhibit different propensities to bind less accessible chromatin. High Hox binding selectivity is also associated with high affinity binding regions, leading to a model where Hox proteins derive binding selectivity through an affinity-based competition with nucleosomes. Provision of the Extradenticle/Homothorax cofactors generally leads to an increase in the number of Hox binding regions and promotes the binding to regions in less accessible chromatin, however the provision of these cofactors has little effect on the overall selectivity of Hox targeting.These studies indicate that chromatin accessibility plays a key role in Hox selectivity and we propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of genomic targets for different Hox proteins. We suggest that this mechanism may also be relevant to other transcription factor families.

scholarly journals High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Bogdan Mitran ◽  
Helge Thisgaard ◽  
Ulrika Rosenström ◽  
Johan Hygum Dam ◽  
Mats Larhed ◽  
...  
Keyword(s):  
Binding Specificity ◽  
Hepatic Uptake ◽  
High Contrast ◽  
Agent Based ◽  
Pet Ct ◽  
Rapid Clearance ◽  
Grpr Antagonist ◽  
High Yields

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied. NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi). The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors.

scholarly journals Switching metalloporphyrin binding specificity of a b-type cytochrome to fluorogenic zinc by design

2019 ◽  
Author(s):  
B. J. Bowen ◽  
A. R. McGarrity ◽  
J-Y. A. Szeto ◽  
C. R. Pudney ◽  
D. D. Jones
Keyword(s):  
Design Process ◽  
Protoporphyrin Ix ◽  
Binding Specificity ◽  
Zinc Protoporphyrin ◽  
Iterative Design ◽  
New Variant

AbstractMetalloporphyrins play important roles in areas ranging from biology to nanoscience. Biology uses a narrow set of metal centres comprising mainly of iron and magnesium. Here, we convert metalloporphyrin specificity of cytochrome b562 from iron (haem) to fluorogenic zinc protoporphyrin IX (ZnPP). Through a computationally guided iterative design process, a variant with a near total preference for ZnPP was generated representing a switch in specificity. The new variant greatly enhanced (≥60 fold) the negligible aqueous fluorescence of free ZnPP in vitro and in vivo.Abstract Figure

scholarly journals Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes

2020 ◽  
Vol 48 (9) ◽  
pp. 5037-5053 ◽  
Author(s):  
Liyang Zhang ◽  
H Tomas Rube ◽  
Christopher A Vakulskas ◽  
Mark A Behlke ◽  
Harmen J Bussemaker ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Specificity ◽  
In Vitro Assays ◽  
Target Discrimination ◽  
Dna Binding Specificity ◽  
Affinity Cleavage ◽  
Target Activity ◽  
Insight Into

Abstract CRISPR RNA-guided endonucleases (RGEs) cut or direct activities to specific genomic loci, yet each has off-target activities that are often unpredictable. We developed a pair of simple in vitro assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq) and cleavage efficiency of RGEs. By separately quantifying binding and cleavage specificity, Spec/SEAM-seq provides detailed mechanistic insight into off-target activity. Feature-based models generated from Spec/SEAM-seq data for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. Together, these applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs, Cas::gRNA pairs, and gain insight into the mechanism and thermodynamics of target discrimination.

scholarly journals Binding of the Vestigial co-factor switches the DNA-target selectivity of the Scalloped selector protein

Development ◽  
2001 ◽  
Vol 128 (17) ◽  
pp. 3295-3305 ◽  
Author(s):  
Georg Halder ◽  
Sean B. Carroll
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Binding Specificity ◽  
General Mechanism ◽  
Binding Selectivity ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Selector Protein

The formation and identity of organs and appendages are regulated by specific selector genes that encode transcription factors that regulate potentially large sets of target genes. The DNA-binding domains of selector proteins often exhibit relatively low DNA-binding specificity in vitro. It is not understood how the target selectivity of most selector proteins is determined in vivo. The Scalloped selector protein controls wing development in Drosophila by regulating the expression of numerous target genes and forming a complex with the Vestigial protein. We show that binding of Vestigial to Scalloped switches the DNA-binding selectivity of Scalloped. Two conserved domains of the Vestigial protein that are not required for Scalloped binding in solution are required for the formation of the heterotetrameric Vestigial-Scalloped complex on DNA. We suggest that Vestigial affects the conformation of Scalloped to create a wing cell-specific DNA-binding selectivity. The modification of selector protein DNA-binding specificity by co-factors appears to be a general mechanism for regulating their target selectivity in vivo.

scholarly journals Direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping (CasKAS)

2021 ◽  
Author(s):  
Georgi K Marinov ◽  
Samuel H. Kim ◽  
S. Tansu Bagdatli ◽  
Alexandro E. Trevino ◽  
Josh Tycko ◽  
...  
Keyword(s):  
Dna Structure ◽  
Binding Specificity ◽  
Practical Application ◽  
Enzyme Binding ◽  
Epigenome Editing ◽  
Genome Wide ◽  
Cas9 Protein ◽  
Target Activity

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structure formed upon binding of a sgRNA-loaded Cas9 protein (''CasKAS''). We demonstrate this method in both in vitro and in vivo contexts.

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