These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair. Abstract Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses.
Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage.
Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological lidase és prostatitis, herein we present the most widely used protocols in lidase és prostatitis field of DNA repair, Fluorescence Immunostaining Lidase és prostatitis and Chromatin Immunoprecipitation ChIPwhich in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively.
These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as lidase és prostatitis post-translational modifications necessary for their fine-tune regulation during DNA repair.
Introduction Log in or Start trial to access full content. These assaults lidase és prostatitis derive from environmental sources, such as UV light or irradiation, as well as from endogenous sources, such as metabolic by-products caused by oxidative stress or replication errors1,2.
These lesions can affect the integrity of either one or both DNA strands, and if the generated errors become persistent, it frequently leads to translocations and genome instability, which may result in tumorigenesis3,4.
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
To maintain genome integrity, multiple repair systems have been developed during evolution. According to the chemical and physical properties of specific types of DNA damage, multiple repair mechanisms can be activated. Mismatches, abasic sites, single-strand breaks, and 8-oxoguanine 8-oxoG can be removed either by mismatch repair or base-excision repair pathway5,6.
Regarding the cell cycle phase, following DNA double-strand break induction, two sub-pathways can be activated: non-homologous end joining NHEJ and homologous recombination HR 1,9.
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NHEJ, which is the dominant pathway in lidase és prostatitis cells, can be activated in all cell cycle phases, representing a faster but error-prone pathway On the other hand, HR is an error-free pathway, in which the DSBs are repaired based on sequence-homology search of lidase és prostatitis sister chromatids, therefore it is mainly present in S and G2 cell cycle phases Therefore, MMEJ is considered to be error-prone and highly mutagenic The DDR is activated as a response to the recruitment and extensive spreading of initiator key players of the repair process around the lesions, contributing to the formation of a repair focus.
This early event is responsible for the recruitment of additional repair factors and the initiation lidase és prostatitis downstream repair processes. Although the exact function of the recruited proteins at the repair focus has not yet been fully characterized, the formation and the dynamics of repair foci have been investigated by several laboratories.
These markers are extensively used to follow the repair kinetics, but their precise role during the repair process remains elusive. Due to the great importance yet poor understanding of DNA repair-related cellular processes, several methods have been developed so far to induce and visualize the DDR. Various methods and systems have been established to induce the desired type of DNA damage.
Here, we focus on the endonuclease-based techniques currently used to study the DDR in mammalian and yeast cells. Aside from highlighting the principles of these techniques, we emphasize both their advantages and disadvantages.
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Protocol Log in or Start trial to access full content. Aspirate the medium and wash the cells with 1x PBS. When the cells detach, stop the trypsin activity by adding culture medium to the cells, yielding a cell suspension. Count the cells using a cell counting chamber. Incubate the cells with the NCS-containing medium for 15 min, then wash them with 1x PBS and add fresh, supplemented culture medium to the cells. Otherwise, use appropriate agent i.
Each incubation and washing step except the antibody incubation should be performed on an orbital shaker with gentle agitation. Following DSB induction and incubation of cells, remove the medium from the attached cells and wash the cells once with 1x PBS.
Permeabilization of cells Remove the fixing solution and wash cells three times with 1x PBS for 5 min each. Remove the PBS and add 0. Incubate the samples for 20 min. Blocking of non-specific binding sites Wash the cells three times with 1x PBS.
Immunofluorescence staining Add the proper amount of primary antibody i. Place each coverslip upside down onto a paraffin film over a 10 µL droplet of the lidase és prostatitis anti-γH2AX antibody. Incubate the samples in a humidity chamber for 1. Place the coverslips back being side up into the well plate and wash three times for 5 min with 1x PBS.
Place each coverslip upside down onto a paraffin lidase és prostatitis over a 10 µL droplet lidase és prostatitis the diluted antibody. Incubate the samples in a humidity chamber at 4 °C for 1 h. Before removing the last PBS washing solution, gently take out the coverslips using a tweezer and needle and then place them upside down onto glass slides with droplets of mounting medium supplemented with DAPI.
When the mounting medium dries, it is recommended to seal the edges of the coverslips with nail polish to prevent shriveling of the samples.
Remove the lidase és prostatitis medium and wash the cells twice with ice-cold 1x PBS. NOTE: Formaldehyde is volatile; always prepare a fresh working solution. In some cases, the formaldehyde solution contains methanol to stabilize it, but it is better to use a methanol-free solution to avoid interference with downstream reactions. Stop lidase és prostatitis fixation with mM glycine and incubate on an orbital shaker with gentle agitation for 5 min at 25 °C.
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Place the plates on ice and wash twice with ice-cold 1x PBS. Scrape the cells in ice-cold 1x PBS and transfer them lidase és prostatitis 15 mL conical tubes. Centrifuge the cells at 2, x g for 5 min at 4 °C. Centrifuge the cell suspension at 2, lidase és prostatitis g for 5 min at 4 °C. Carefully discard the supernatant and resuspend the pellet inµL of nuclear lysis lidase és prostatitis 50 mM Tris-HCl pH 8.
Transfer the lysate into a polystyrene conical tube suitable for sonication. The solution should turn transparent following sonication. Sonicate the lysate to shear DNA to an average fragment size ofbp. NOTE: The appropriate sonication cycles and conditions should be set according to the cell type and the sonication equipment. Reversal of crosslinking, determination of sonicated fragment sizes Take out µL of the sonicated sample to verify the fragment size of the sonicated chromatin.
Add 0. Incubate the samples at 65 °C overnight. Vortex for 1 min. Centrifuge at 13, x g for 10 min. Transfer the upper aqueous phase to a new microcentrifuge tube. Add 1 volume chloroform-isoamyl alcohol mix to each sample. Add 2. Centrifuge the samples at 13, x g for 10 min at 4 °C. Remove the ethanol and air dry the pellet. Resuspend the pellet in 10 µL of TE. Run the samples on a 0. The sonicated chromatin size should be around bp. NOTE: Use bromophenol blue-free loading buffer because the size of this dye is approximately bp, which can disturb the proper detection of chromatin fragments.
Instead, it is recommended to use loading buffer complemented with xylene-cyanole, which is approximately 3, bp. If the chromatin size is acceptable, dilute the frozen chromatin samples from step 2. Preparation of beads, pre-clearing, and immunoprecipitation Prepare beads sheep anti-rabbit or mouse IgG for the pre-clearing and immunoprecipitation steps. Resuspend the beads in the same volume of RIPA buffer as in step 2.
Recept a chaga- hoz a prosztatitisből µg chromatin of each sample with 4 µL of the beads via rotation for h at 4 °C. TICs only require a final volume of up to µL. Precipitate the beads with a magnet and transfer the supernatant to a new microcentrifuge tube.
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- Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage | Protocol
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Next day, add 40 µL of washed beads to each sample except TIC and incubate them overnight, rotating at 4 °C. Transfer the supernatant to a new tube and elute beads again in µL of Elution buffer. Combine eluates µL final volume. Add NaCl to a final concentration of mM in each sample. Incubate the samples at 65 °C without shaking for at least 6 h.
The next day, centrifuge for 30 min at 13, x g at 4 °C.
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Discard the supernatant and air dry the pellets. Resuspend the pellets in µL of TE and add 0. Incubate at 37 °C for 20 min to activate the RNase. Instead, it is recommended to use a commercially available kit see Table of Materials. Add 1 volume of chloroform-isoamyl alcohol mix to each sample. DNA extraction Add 2. Resuspend the pellet in 50 µL of TE. Representative Results Log in or Start trial to access full content.
However, it should be noted that stable transfection ensures a lidase és prostatitis cell population, which gives a unified and thus more reliable cellular response. In the case of transient transfection, only a small proportion of the cell population takes up and maintains the plasmid, which introduces diversity into the experiment.
For transfection, commercially available transfection reagents or viral infection-based methods can also be used. If a microscopic visualization technique is to be applied and transient transfection is lidase és prostatitis, the directed DSBs can be induced by 4-OHT addition 24 h after transfection, which binds to the ER-fused endonucleases and allows the nuclear translocation and DSB induction.
To determine the most appropriate time-points, immunofluorescence-based microscopy and western blot detection of γH2AX at different time-points following 4-OHT treatment can be performed. Under physiological conditions, a maximum of γH2AX foci per cell can be detected, and the formation of strong repair foci can be triggered by endonucleases or various other techniques not discussed here e. For this reason, representative time-points at 0 h, 30 min, 1, 2, 4, and 8 h following 4-OHT treatments are recommended.
To track Előrehaladott prosztatagyulladás repair processes, the most commonly used cell line is U2OS, as all known repair pathways are fully functional in these cells. When investigating several proteins in the same cells, co-localization can be studied by combining antibodies conjugated with different fluorophores with different emission wavelengths raised in different animal species prostatitis krónikus a levtől shown in Figure 1.
Incubation for 4 h with 4-OHT, 24 h after doxycycline addition, can induce high number of DSBs since the endonuclease has been translocated to the nucleus Figure 1. Scale bars represent 20 µm. Please click here to view a larger version of this figure. Co-localization of repair proteins at the damage site indicates that they are recruited to the same DNA lesion site, but they do not necessarily interact with each other.
The resolution of the confocal microscopy is approximately nm; to determine the binding pattern of specific repair proteins at the break site, super-resolution microscopy STORM is instead recommended However, this method requires expensive microscopic equipment and an expert researcher.
Upon 4-OHT addition, both endonucleases can cut the DNA in a sequence specific manner which provides us the opportunity to design locus specific primers to the expected break sites and their surrounding genomic regions. By applying γH2AX antibody in the immunoprecipitation part of the ChIP, we can temporally follow the DNA repair kinetics upon different conditions such as silencing or inhibition of certain repair factors of interest, i.
On the left part of the image, the timely lidase és prostatitis γH2AX signal is shown at the break site while on the right part, the γH2AX distribution is represented at a control gene region at which DSBs have not been induced.
The represented results are derived from one biological experiment, and error bars indicate the variations of the corresponding sample lidase és prostatitis. Discussion Log in or Start trial to access full content. Preserving genetic information is crucial for cells since mutations occurring in genes involved in repair processes are among the leading causes of tumorigenesis and therefore elucidating the key steps of DNA repair pathways is essential.
Biochemical techniques i. Performing ChIP experiments is laboring, troublesome and many considerations have to be taken into account when designing specific experiment to study the process of DSB repair.
Since this method reduces the yield of recovered DNA, our personal advice is to rather use specific DNA-purification kit. When all critical points have been properly addressed, ChIP can gyertya prosztatitis kezelési diagram valuable data for the occupancy of the desired protein at different genomic loci and can unravel critical steps in DNA repair processes.
However, ChIP combined with qPCR is an indirect approach to study the protein distribution at selected genomic regions and does not allow to specifically recognize the DNA binding site or directly examine the function of the protein. Mono- or polyclonal antibodies used to capture the protein-DNA complexes can also cross-react lidase és prostatitis other proteins leading to false-positive data and therefore, the antibodies used in this technique should be ChIP-grade and highly specific against the protein of interest.
The former one relies on hybridizing the immunoprecipitated and purified DNA fragments on a microarray with a large variety of small random DNA sequences which further amplifies the annealed sequences providing valuable information about protein binding sites.
However, ChIP-seq has emerged as an attractive alternative approach since it provides genome-wide mapping of protein-DNA complexes with higher resolution than ChIP-on-chip and high-throughput genome sequencing.
ChIP-seq has revolutionized the field of DNA repair by disclosing DNA binding sites of various transcription factors providing insights into the gene regulation and unravelling chromatin landscapes in a genome-wide scale According to this, the field of DNA repair has tremendously profited from ChIP-seq since these data play lidase és prostatitis crucial role in various diseases and biological pathways, such as cancer progression.
Nonetheless, various modifications of the ChIP method have been developed such as HaloChIP, which does not require specific antibody against the protein of interest but rather uses sequences encoding DNA-binding proteins fused with HaloTag, which are transfected to cells and subsequently to crosslinking, the desired protein-DNA complexes can be captured lidase és prostatitis using HaloLink Resin. However, this technique relies on overexpression and not the endogenous level of the desired proteins which kezelés prosztatitis herpesz result in misinterpreted data Furthermore, microscopic techniques provide valuable information about the spatiotemporal tracking of DNA damage repair, even in a single-cell level.