1 Protein-DNA interakce Protein-DNA interaction F1MG1_Metods of MB PDB id: 5KKQ, Hashimoto Mgr. Denis Šubert Mgr. Marie Brázdová, Ph.D. https://www.researchgate.net/f igure/Mutant-p53-proteins- carry-out-novel-oncogenic- interactions-A-Signaling-in-the- p53_fig2_277087873 1 Protein-DNA interactions 2 Protein-DNA interakce —Screening of interaction partners —Localization of the interaction within the chromatin —Sequential structural preference —Protein function (TRF, Helicases, Chromatin, DNA-repair) • A wide range of biophysical chemistry methods have been used to study interactions between proteins and nucleic acids. • particularly good for determining the strength (affinity) of interactions • High affinity, μM–nM: tend to involve sequential interactions, • Low affinity, mM – μM: proteins tend to recognize aspects of the "whole" structure, ie. MB2024-DNA protein interactions DNA-protein binding motifs 3 Protein-DNA interakce Hydrogen bonds: Adenine - Gln/ Asn Guanine - Arg Salt bridges: Phosphate residue – Arg/ Lys Through coordinately bonded metals Zinc finger motif – Zn2+ Gln/Asn forms specific H-bonds with N-6 H-7 H of Adenine Arg forms specific bonds with the Cytosine-Guanine pair MB2024-DNA protein interactions The most common DNA-binding motifs HTH Zinc-Finger Leucin zip Binding to DNA major groove DNA major groove DNA major groove Composition Helix-turn-Helix ß-sheet-ß-sheet-Helix Helix-Helix Structure 4 Protein-DNA interakceMB2024-DNA protein interactions MB2024-DNA protein interactions5 MB2024-DNA protein interactions 6 Methods for studying DNA-protein interactions - Chip seq Pull-down assay EMSA FRET ITC Co-crystallization NMR A yeast two-hybrid system Gene expression SELEX database (interactome and complexes...) genetic methods MB2024-DNA protein interactions 7 DNA-protein interakce 8 (např. ChIP = Chromatin Immunoprecipitation) Detection of DNA/RNA binding sites ChIP (chromatinová imunoprecipitace) MB2024-DNA protein interactions Image credit: Song, C., Zhang, S., and Huang, H. (2015). Choosing a suitable method for the identification of replication origins in microbial genomes. Front. Microbiol. Chromatin immunoprecipitation (ChIP) is a technique that determines whether a protein of interest interacts with a specific DNA sequence. This technique is often used to study the repertoire of sites on DNA that are bound by specific transcription factors or histone proteins and to look at the precise genomic locations of various histone modifications (including acetylation, phosphorylation, or methylation). ChIP- seq ChIP-cloning ChIP on ChIP MB2024-DNA protein interactions9 SELEX elute clone & sequence C.Tuerk, L. Gold Systematic evolution of high-affinity RNA ligands of bacteriophage T4 DNA polymerase in vitro. Science 249:505-510 (1990). Random oligonucleotide pool Affinity matrix Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers.[1][2][3] Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB (selected and amplified binding site) and CASTing (cyclic amplification and selection of targets)[4][5] SELEX was first introduced in 1990. In 2015 a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the SELEX discovery.[6] MB2024-DNA protein interactions10 11 Imunoprecipitation-protein -DNA (DNA), pull down - method of isolating specific proteins from mixtures (lysates, purified...) using antibodies - antibodies in a complex with their antigens are separated from other molecules using proteins A or G (source of bacteria), which bind immunoglobulins and at the same time are immobilized on a solid surface (BEADS) - proteins A and G bind to the Fc region of heavy chains the Fab region is still available for antigen binding DNA precipitation MB2024-DNA protein interactions MB2024-DNA protein interactions12 Imunoprecipitation protein–DNA - imobilisation - method of isolating specific proteins from mixtures (lysates, purified...) by means of DNA-bound to beads - protein precipitation - WB detection MB2024-DNA protein interactions https://www.sinobiological.com/resource/protein- review/affinity-tag 13 Different systems of affinity tags and their interaction partners used for protein purification Protein-DNA Footprinting MB2024-DNA protein interactions 14 - Used to identify the target region of interaction within the DNA sequence ̶One strand of DNA must be labeled (Radioactive/fluorescent) ̶Uses DNase I enzyme or chemical cleavage (piperidine) ̶Areas where interaction occurs are protected from cleavage – missing stripes (bands) assumption: protein-bound DNA will be protected from chemical cleavage at the binding site. Isolate the DNA fragment that contains the binding site and "label". Bind the protein to the DNA in one tube; keep the other as a "naked DNA" control Treat both samples with a chemical or enzymatic agent. Separate the fragments by gel electrophoresis and visualize the bands on X-ray film or an imaging plate "Footprinting" is a technique to identify the DNA-binding site Footprinting Results of RNA Polymerase Bound to Promoter MB2024-DNA protein interactions 15 Chemic cleavage: AG: Formic acid CT:Hydrazine C:Hydrazine+NaCl G:Dimethylsulphate -piperidinu DNA/RNA footprinting MB2024-DNA protein interactions16 Yeast two-hybrid systems Y2H ̶ a molecular biology method commonly used to study interactions between proteins. ̶ The functioning of Y2H is made possible by the fact that transcription factors have a modular structure and these module-domains can function separately or in fusion with another protein. ̶ In the most common form of Y2H, the yeast transcription factor Gal4 is split in this way. Its DNA binding domain is fused to one protein ("bait"), usually known, whose binding partners Y2H will search for, while the activation domain of Gal4 is fused to a protein ("prey") that is a potential binding partner of the investigated protein . ̶ The interaction between "bait" and "prey" restores the original function of Gal4, which subsequently transactivates the relevant reporter genes. Due to its versatility and simplicity, Y2H can be used to rapidly analyze large cDNA libraries encoding proteins fused to the Gal4 DNA binding domain. However, Y2H results must be verified by other methods, as they are usually loaded with a significant error, either false negative or false positive results. ̶ The yeast two-hybrid system, its possible uses, variations and limitations have recently been described in great detail (Brückner et al. 2009). Report gene: lacZ (B-galaktosidase) Luciferase GFP MB2024-DNA protein interactions17 V nejběžnější podobě Y2H je takto rozdělen kvasinkový transkripční faktor Gal4. Jeho DNA vazebná doména (BD) je fúzována s jedním proteinem (bait – „návnada“), obvykle známým, jehož vazebné partnery bude Y2H vyhledávat, zatímco aktivační doména (AD) Gal4 je fúzována s proteinem (prey – „kořist“), který je potenciální vazebný partner zkoumaného proteinu. https://cs.wikipedia.org/wiki/Dvouhybridový_systém Aplikace/Využití: - protein-protein interakce - DNA-protein interakce - Analýza genové exprese- regulace - reporterový test (DNA vazebné elementy, DNA regulační elementy) Geny nebo markery reportéra poskytují vhodný prostředek identifikovat a analyzovat regulační prvky genů. Systém reportérů měří transkripční činnost (interakce cisprvků na předkladatele s předkladatelem trans-působící faktory). MB2024-DNA protein interactions18 http://photobiology.info/Ohmiya.html Dual-Luciferase® Reporter (DLR™) Assay The Dual-Luciferase® Reporter (DLR™) Assay System(a–c) provides an efficient means of performing dual-reporter assays. In the DLR™ Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. MB2024-DNA protein interactions19 Luciferase Reporter Assay ̶ The Dual-Luciferase® Reporter (DLR™) Assay System(a–c) provides an efficient means of performing dual-reporter assays. In the DLR™ Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. MB2024-DNA protein interactions20 FRET Förster/fluorescence resonance energy transfer Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other.[5] Such measurements are used as a research tool in fields including biology and chemistry. is a mechanism describing energy transfer between two light-sensitive molecules (chromophores).[1] A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole– dipole coupling Förster/fluorescence resonance energy transferFRET MB2024-DNA protein interactions 21 Primary Conditions for FRET •Donor and acceptor molecules must be in close proximity (typically 10–100 Å). •The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor (Figure 1). •Donor and acceptor transition dipole orientations must be approximately parallel. MB2024-DNA protein interactions22 MB2024-DNA protein interactions23 MB2024-DNA protein interactions24 Determination of protein-DNA interaction affinity ̶ The interaction takes place in vitro ̶ Necessity of DNA labeling (Radioactivity/fluorescence) ̶ Separation by gel electrophoresis (PA, Agarose) ̶ We monitor the retardation (shift) of the migration of the DNA-protein complex in the electric field ̶ The migration of a molecule (complex) in an electric field depends on its size and charge EMSA (“Gel Shift” Assay) • Electrophoretic Mobility Shift Assay (EMSA) or “gel shift” can provide information about protein-NA interactions MB2024-DNA protein interactions [Protein] DNA + protein +Ab DNA + protein + Ab M DNA DNA alone Poměrně přímočará technika, ale poskytuje pouze přesvědčivá data pro interakce s vysokou afinitou (typicky <μM) TEST 25 p53CD p53b p53 (ng) 250 500 1000 125 250 500 I p53CON p53CD+CON p53CON TAT triplex p53b+CON 1 2 3 4 5 6 7 8 9 10 11 12 13 14 250 500 1000 125 250 500 I p53CD p53b TAT triplex p53b+TAT p53CD p53b p53 (ng) 250 500 1000 125 250 500 I p53CD p53b p53 (ng) 250 500 1000 125 250 500 I p53CD p53b p53 (ng) 250 500 1000 125 250 500 I p53 (ng) 250 500 1000 125 250 500 I p53CON p53CD+CON p53CON TAT triplex p53b+CON 1 2 3 4 5 6 7 8 9 10 11 12 13 14 250 500 1000 125 250 500 I p53CD p53b TAT triplex p53b+TAT CD x C-terminus MB2024-DNA protein interactions26 MB2024-DNA protein interactions27 MB2024-DNA protein interactions28 NMR Co-crystalisation NMR Summary: gene expression analysis - study of transcription (mRNA-northern transfer, RT-PCR, IN SITU hybridization, primer extension) comparison of transcriptomes (RT-PCR, siRNA, DNA microarrays, ..) Analysis of promoters and protein-DNA interactions (reporter genes, promoter localization, identification of promoter regulatory regionselements, footprinting, EMSA...) Analysis of translation (proteins-WB, ChIP, IP) MB2024-DNA protein interactions29