scholarly journals Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexandra Stützer ◽  
Luisa M. Welp ◽  
Monika Raabe ◽  
Timo Sachsenberg ◽  
Christin Kappert ◽  
...  
Keyword(s):  
Uv Light ◽  
Amino Acid Level ◽  
Dna Interaction ◽  
Cross Linking ◽  
Dna Interactions ◽  
Single Experiment ◽  
Protein Dna Interactions ◽  
Nucleosome Binding ◽  
Binding Interfaces

Abstract Protein–DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein–DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein–RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.

scholarly journals In vivo protein-DNA interactions at the c-jun promoter: preformed complexes mediate the UV response.

1993 ◽  
Vol 13 (9) ◽  
pp. 5490-5499 ◽  
Author(s):  
D Rozek ◽  
G P Pfeifer
Keyword(s):  
Cpg Island ◽  
Uv Light ◽  
Dimethyl Sulfate ◽  
Related Factor ◽  
Dna Interactions ◽  
Intact Cells ◽  
Response Elements ◽  
Protein Dna Interactions ◽  
Uv Response

Irradiation of cells with UV light triggers a genetic response, called the UV response, which results in induction of a set of genes containing AP-1-binding sites. The c-jun gene itself, which codes for AP-1-binding activity, is strongly (> 100-fold) and rapidly activated by UV. The UV induction of c-jun is mediated by two UV response elements consisting of AP-1-like sequences within its 5' control region. We have analyzed protein-DNA interactions in vivo at the c-jun promoter in noninduced and UV-irradiated HeLa cells. In vivo footprint analysis was performed by using dimethyl sulfate on intact cells and DNase I on lysolecithihin-permeabilized cells in conjunction with ligation-mediated polymerase chain reaction to cover about 450 bp of the c-jun promoter, including the transcription start sites. We find that this region does not contain methylated cytosines and is thus a typical CpG island. In uninduced cells, in vivo protein-DNA interactions were localized to an AP-1-like sequence (nucleotides [nt] -71 to -64), a CCAAT box element (nt -91 to -87), two SP1 sequences (nt -115 to -110 and -123 to -118), a nuclear factor jun site (nt -140 to -132), and a second AP-1-like sequence (nt -190 to -183). These results indicate that complex protein-DNA interactions exist at the c-jun promoter prior to induction by an external stimulus. Surprisingly, after stimulation of c-jun expression by UV irradiation, all in vivo protein-DNA contacts remained essentially unchanged, including the two UV response elements located at the AP-1-like sequences. The UV-induced signalling cascade leads to phosphorylation of c-Jun on serines 63 and 73 (Y. Devary, R.A. Gottlieb, T. Smeal, and M. Karin, Cell 71:1081-1091, 1992). Taken together, these data suggest that modification of the transactivating domain of DNA-bound c-Jun or a closely related factor may trigger the rapid induction of the c-jun gene.

Diazirine-Based DNA Photo-Cross-Linking Probes for the Study of Protein–DNA Interactions

2008 ◽  
Vol 120 (1) ◽  
pp. 96-99 ◽  
Author(s):  
Uddhav Kumar Shigdel ◽  
Junliang Zhang ◽  
Chuan He
Keyword(s):  
Cross Linking ◽  
Dna Interactions ◽  
Protein Dna Interactions

scholarly journals DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Applied Nano ◽  
2022 ◽  
Vol 3 (1) ◽  
pp. 16-41
Author(s):  
Aurimas Kopūstas ◽  
Mindaugas Zaremba ◽  
Marijonas Tutkus
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Dna Interaction ◽  
Dna Interactions ◽  
Ensemble Averaging ◽  
Target Search ◽  
Single Molecule Level ◽  
Protein Dna Interactions ◽  
Long Time

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

In vivo protein-DNA interactions at the c-jun promoter: preformed complexes mediate the UV response

1993 ◽  
Vol 13 (9) ◽  
pp. 5490-5499
Author(s):  
D Rozek ◽  
G P Pfeifer
Keyword(s):  
Cpg Island ◽  
Uv Light ◽  
Dimethyl Sulfate ◽  
Related Factor ◽  
Dna Interactions ◽  
Intact Cells ◽  
Response Elements ◽  
Protein Dna Interactions ◽  
Uv Response

Irradiation of cells with UV light triggers a genetic response, called the UV response, which results in induction of a set of genes containing AP-1-binding sites. The c-jun gene itself, which codes for AP-1-binding activity, is strongly (> 100-fold) and rapidly activated by UV. The UV induction of c-jun is mediated by two UV response elements consisting of AP-1-like sequences within its 5' control region. We have analyzed protein-DNA interactions in vivo at the c-jun promoter in noninduced and UV-irradiated HeLa cells. In vivo footprint analysis was performed by using dimethyl sulfate on intact cells and DNase I on lysolecithihin-permeabilized cells in conjunction with ligation-mediated polymerase chain reaction to cover about 450 bp of the c-jun promoter, including the transcription start sites. We find that this region does not contain methylated cytosines and is thus a typical CpG island. In uninduced cells, in vivo protein-DNA interactions were localized to an AP-1-like sequence (nucleotides [nt] -71 to -64), a CCAAT box element (nt -91 to -87), two SP1 sequences (nt -115 to -110 and -123 to -118), a nuclear factor jun site (nt -140 to -132), and a second AP-1-like sequence (nt -190 to -183). These results indicate that complex protein-DNA interactions exist at the c-jun promoter prior to induction by an external stimulus. Surprisingly, after stimulation of c-jun expression by UV irradiation, all in vivo protein-DNA contacts remained essentially unchanged, including the two UV response elements located at the AP-1-like sequences. The UV-induced signalling cascade leads to phosphorylation of c-Jun on serines 63 and 73 (Y. Devary, R.A. Gottlieb, T. Smeal, and M. Karin, Cell 71:1081-1091, 1992). Taken together, these data suggest that modification of the transactivating domain of DNA-bound c-Jun or a closely related factor may trigger the rapid induction of the c-jun gene.

Dynamical perspective of protein-DNA interaction

2014 ◽  
Vol 5 (1) ◽  
pp. 21-43 ◽  
Author(s):  
Subrata Batabyal ◽  
Susobhan Choudhury ◽  
Dilip Sao ◽  
Tanumoy Mondol ◽  
Samir Kumar Pal
Keyword(s):  
Biological Activities ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Interaction ◽  
Specific Protein ◽  
Water Dynamics ◽  
Dna Interactions ◽  
Terminal Domain ◽  
Protein Dna Interactions ◽  
Dna Complex

AbstractThe interactions between protein-DNA are essential for various biological activities. In this review, we provide an overview of protein-DNA interactions that emphasizes the importance of dynamical aspects. We divide protein-DNA interactions into two categories: nonspecific and specific and both the categories would be discussed highlighting some of our relevant work. In the case of nonspecific protein-DNA interaction, solvation studies (picosecond and femtosecond-resolved) explore the role environmental dynamics and change in the micropolarity around DNA molecules upon complexation with histone protein (H1). While exploring the specific protein-DNA interaction at λ-repressor-operator sites interaction, particularly OR1 and OR2, it was observed that the interfacial water dynamics is minimally perturbed upon interaction with DNA, suggesting the labile interface in the protein-DNA complex. Förster resonance energy transfer (FRET) study revealed that the structure of the protein is more compact in repressor-OR2 complex than in the repressor-OR1 complex. Fluorescence anisotropy studies indicated enhanced flexibility of the C-terminal domain of the repressor at fast timescales after complex formation with OR1. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with OR1 DNA compared to OR2 DNA were found to have pronounced effect on the rate of photoinduced electron transfer.

scholarly journals Sharing DNA-binding information across structurally similar proteins enables accurate specificity determination

2019 ◽  
Vol 48 (2) ◽  
pp. e9-e9
Author(s):  
Joshua L Wetzel ◽  
Mona Singh
Keyword(s):  
Dna Binding ◽  
Zinc Finger Protein ◽  
Dna Interaction ◽  
Label Propagation ◽  
Structural Domain ◽  
Dna Interactions ◽  
Interaction Information ◽  
Protein Dna Interactions ◽  
Powerful Means ◽  
The Individual

Abstract We are now in an era where protein–DNA interactions have been experimentally assayed for thousands of DNA-binding proteins. In order to infer DNA-binding specificities from these data, numerous sophisticated computational methods have been developed. These approaches typically infer DNA-binding specificities by considering interactions for each protein independently, ignoring related and potentially valuable interaction information across other proteins that bind DNA via the same structural domain. Here we introduce a framework for inferring DNA-binding specificities by considering protein–DNA interactions for entire groups of structurally similar proteins simultaneously. We devise both constrained optimization and label propagation algorithms for this task, each balancing observations at the individual protein level against dataset-wide consistency of interaction preferences. We test our approaches on two large, independent Cys2His2 zinc finger protein–DNA interaction datasets. We demonstrate that jointly inferring specificities within each dataset individually dramatically improves accuracy, leading to increased agreement both between these two datasets and with a fixed external standard. Overall, our results suggest that sharing protein–DNA interaction information across structurally similar proteins is a powerful means to enable accurate inference of DNA-binding specificities.

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