DNA Repair - Science topic

Dear fellow ResearchGate members ^^,

I have a question regarding the Protein DNA-PKcs from the NHEJ repair Pathway.

This protein has a subdomain called FBR Region, which I think was identified by being homologous to the according FBR Region in mTOR where Rapamycin binds.

I was wondering (since I can't really find any papers about it) if something is known about small molecules binding to that region in DNA-PKcs.

Thanks for your help.

Best regards,

Ben

Hello, I'm preparing libraries for Illumina sequencing and i would like to know if I have to do DNA end repair immediately after shearing, or if I can freeze sheared DNA and do end repair a week or two later. Thanks

This question is spurred by the different effects of BRCA1/2 vs. PARP.

Thanks in advance for insights.

I want to simulate a system of protein and DNA in the context of a DNA repair mechanism. So, I would want the average protein concentration of the DNA repair protein in the cell.

ABSTRACT, study 1:

Novel coronavirus disease 2019 (COVID-19) is the biggest threat to human being globally. The first case was identified in a patient with flu symptoms along with severe acute respiratory syndrome in Wuhan, China in December 2019 and now it has spread in more than 200 countries. COVID-19 is more lethal in the elderly and people with an underlying condition such as asthma, cancer, diabetes. Here we performed bioinformatic analysis to investigate the interaction of S2 subunit protein of SARS-nCoV-2 of novel coronavirus with tumor suppressor proteins p53 and BRCA-1/2. In this short communication we report the interaction between S2 subunit proteins with tumor suppressor proteins for the first time. This preliminary result will open up a new direction to investigate the effect of a novel coronavirus in cancer patients.

ABSTRACT, study 2:

Here, by using an in vitro cell line, we report that the SARS-CoV-2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.

What next?

DNA Damage Question

I am performing Phospho-yH2AX immunofluorescent staining of cells treated with either 0.1% DMSO or a drug inhibitor in order to measure DNA damage. Unexpectedly, I am observing high levels of signal in the DMSO treated cells. The cells are grown on Chambered Cell Culture Slides from CellTreat. It does not appear to be cell type specific given I have observed this with 5 breast cancer cell lines and 2 prostate cancer lines. I am using the CST Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718 antibody in Normal Donkey Serum at the recommend 1:400 dilution, so I don't believe its non-specific signal either. Has anyone encountered this issue before or know of any alternative explanations.

For reference I have attached an image below where H2AX signal is shown in pink these are completely untreated suggesting it is not an effect of DMSO

There are so many DNA polymerase and they have many activities like polymerase activity, gap filling, proof reading activity, and DNA repair activities. Which one DNA polymerase does not have DNA repair activity?

It is well known that Fpg and Endonuclease IV can cleave AP site in double strand DNA. But can they cleave AP (Apurinic/apyrimidinic) site in single strand DNA?

As stated as in the title, I am wondering that,

When DNA repair occurs via HDR, is the template donor DNA with homology arms itself integrated or is it only used as a reference sequence that is used for cells to polymerizes new DNA?

Thank you for taking your time to answer me.

I'm doing immunofluorescence assay (in cell western, ICW) in HepG2 cell treated with Heterocyclic Amines. I measure the gamma H2AX foci as endpoint, but I found that γH2AX induction may decline at high concentration (induction lower than low concentration), like the DNA damage have a critical value.

I had found some paper that DNA damage signal decrease may result from dissociation of H2AX histones or chromatin compaction lead to repress transcription, and promote DNA repair and mitotic delay.

Can someone help me? Is that any scientific theorys about this problem.

Thank you

If I have more number of foci, does it represent nuclear damage or nuclear repair? Some cells show pan nuclear staining, does it mean the cell underwent apoptosis?

I am new to this area. Any help would be appreciated.

I am cloning endonuclease in pET vector

I'm treating my cells with a drug inducing SSB rapidly converting into DSB. While I'm able to measure total level of DNA damage via Comet assay and all kinds of yH2AX, is there a specific DSB DNA repair assay? I know there are kits for BER and NER, but not aware of other types. Thanks

Hello everyone!

I am trying to make a library of metagenomic DNA in the plasmid PBlueScript SK+. I fragmented metagenomic DNA by sonication and I confirmed the average size of the fragments is 3kb. I then repaired the DNA fragments with Klenow fragment (NewEngland #EP0054) so that the the ends are blunt. After that, I purified this reapired DNA. Previously, I digested the Plasmid PBScript with EcoRV that also leaves blunt ends. I tried to performed ligation in several Vector:Insert ratios (1:3, 1:1, 3:1...). None of the ratios gave a large number of clones but Ratio 3:1 exhibited the best results in terms of number of colonies and higher proportion of white/blue colonies, which is good .

I extracted plasmid from 15 white colonies of this ligation, I performed Restriction digestion with enzymes XhoI and NotI (that flank EcoRV at both sides in plasmid PBlueScript) so that I could see two bands in agarose gel (1 Plasmid backbone and 2 metagenomic insert). The problem is I obatined a unique band for most of the clones and, moreover, this single band does not match the size of the empty plasmid. Its smaller !!

To me, this result does not make sense. I thought there could be recombination events in the cell but I´m using DH5 alpha strain so it should not happen. Could it be due to an effect of ligation of so long fragments whit blue ends? Am I missing anything?

I have no experience with library construction so any help will be very helpful.

Thanks a lot in advance!

Jorge

I have read many DNA Damage related papers and most of them have used U2-OS cell line for IR with Knock down and Overexpression conditions. Is there any particular reason for using/preferring U2-OS cells, though these processes happen in pretty much every cancer cell line.

p53 is considered as the Guardian of the cell and suppresses tumorigenic genomic rearrangements. I want to learn more that the exact role of p53 in repairing faulty DNA repair mechanism in vivo following transient cell cycle arrest. Any specific sources/articles/papers and materials on this topic would be much appreciated.

I would like to compare the DNA damage at basal level (without any treatment) between a cell line that lacks the expression of one protein vs the parental cell line. I have read that the comet assay is a simple and rapid technique that allow to see if one cell have some defect in DNA repair (single or double breaks) but I am not sure that is the right technique for my experiment or I should search for a different approach.

Any suggestion would be very appreciated.

Thanks!!!!

what are the parameters used to measure the DNA double strand break in cancer cell and DNA repair for it as to know if the drug affect the DNA itself by breaking it or inhibit the DNA repair mechanism for the double strand DNA break

please with references as possible

I am planning to transfect HEK cells with one plasmid expressing Cas9-gRNA and another plasmid expressing luciferase. The Cas9-gRNA will target the luciferase gene on the plasmid. If a double stranded break occurs in the transfected luciferase plasmid, what will be the fate of this plasmid? Will it undergo NHEJ like chromosomal DNA? Or might it be linearised and degraded? Thanks for your help - please direct me to any papers you might be aware of that address this topic if you know of them.

Hi, do you have any recommendation for UV lamp to buy for causing DNA damage in vitro DNA repair comet assay? There are many options so I was wondering if anyone has particularly good experience with a specific product.

Thank you!

In chemopreventive studies (using UV radiation for induce damage) I won't to test the preventive effects of some agents, namely by studying Dna repair. However I would like to use also a positive control...some compound or agent that has preventive effect by inducing DNA repair.

Hello

I would like to conduct a mock experiment on plant cells nucleotide excision repair. the UV sourse I have is a tube light used in laminar hood.

emmission @254nm (85% of total radiationis 254)

lamp output is 13.4W

length : 893mm

diameter : 25.5

Apart from these, i have measured illuminance using illuminometer. the mechine is not really geared for UV radiation but i thought i get a rough estimation. at 30 cm distance from lamp it showed 200 lux.

According to literature, 20 J/square meter of UV irradience is opetimal for viability vs labelling efficiency and i would like to start with the same.

Now the information i needed for starting my experiment is :

I am trying to purify a protein involved in DNA repair and is a part of supra-molecular complex. This protein was over-expressed using pET28a construct in BL21 2DE3 cells but its induction was very less (almost NIL). I switched to codon optimized strain (Rosetta 2DE3) cells which gave me a decent expression but fairly large amount of protein was going in to insoluble fractions. so i used triton X 100 (0.1%) in lysis buffer but it did not make any difference. Finally, use of sarkosyl (0.5 to 1%) gave me very good recovery in the soluble fraction, but then i am not sure whether its use is going to affect any of down stream studies (biophysical and crystallization) I wish to carry out with the purified protein. Can somebody tell me the dos and donts for using sarkosyl in protein purification strategies.

Hi,

I'm a 4th year MD/PhD student from Dr. Hamid Boulares' lab at LSUHSC-New Orleans looking for collaborators with an expertise in DNA damage repair fidelity to help finish our paper with last few experiments.

I need to check the DNA repair fidelity of some samples to complete my paper.

Please message me if you are interested in the collaboration.

Thank you very much!

Hi everybody,

53BP1 is known to block single strand resection which is an essential mechanism for the Homologous Repair DNA pathway, and by consequence, promotes the NHEJ pathway. In BRCA1 mutated cells, Inhibition of 53BP1 favor a reactivation of HR pathway. The consequence is an apparition of PARP inhibitors resistance. My question is, if the inhibition of 53BP1 promotes the HR pathway, why is there a resistance with PARPi (which promote ssDNA breaks and after dsDNA breaks) but seems to improve the IR hypersensitivity of cells (which triggers ssDNA and dsDNA breaks as well) ? I'm really confused about that and I don't if I am enough clear in my question. So don't hesitate to ask me to be more clear :)

Thank you for your help

Dear all,

We have recently performed CRISPR Cas9 knocking out our gene of interest in the U87 glioma cell line. We used the pX459 plasmid from Addgene and tested various gRNAs (always only one type of gRNA at a time). We selected clones based on the absence of the protein product and performed sequencing after subcloning of the PCR product in bacteria. We detected only one type of indel in all four analyzed clones (sequencing 4-5 bacterial colonies for each clone, different indels in different clones). We further directly sequenced the PCR product in 2 of the most promising clones and obtained a clear sequence showing the expected indel in the targeted region. We performed the mismatch-cleavage assay in one of these clones, which showed no cleavage, further supporting that we have the same indel in both alleles.

We are happy to have these nice homozygous knockouts, but it makes me wonder why there are not two different genetic changes in our cells? We confirmed that the cells have 2 alleles of the target gene, to my knowledge the usual type of repair for the double strand breaks should be error prone NHEJ. What is the variability of indels you obtain/would expect after CRISPR Cas9 cleavage in a cancer cell line?

Thanks for your thoughts, Petr.

Can someone please recommend me the protocol for the gamma H2AX foci formation assay?

Many thanks in advance, 

Arun. 

Hi everyone,

I'm new to the Researchgate community. I am using sequencing gels to separate radiolabeled (32P dATP) DNA fragments that are generated during DNA repair. I often encounter smear-like artifacts in empty lanes (that are not loaded with any samples) close to loaded lanes. This prevents me from being able to interpret the data. Does anyone know the cause of this kind of artifact and how It can be prevented? 

I run 0.4 mm thick 7% polyacrylamade gels using the S2 sequencing gel electrophoresis apparatus and shark tooth combs.  

Hello everyone,

why some mutation in very important genes is related to some cancer and not the other. You might say this depends on the gene function on the specific cell. So why mutations in DNA repair genes are associated with specific cancer and not the other?

Dear all,

Does anyone know a publication/ data in plants or yeast on the proportions of DNA repair via NHEJ and HR in different stages of the cell cycle comparable to the findings of Mao et al. from 2008 (DNA Repair 7: 1765-1771 and Cell Cycle 7(18): 2902-2906) in mammalian cells?

Contrary to the common hypothesis (NHEJ in G1 phase, HR in S and G2) their studies show NHEJ as very fast and predominant repair pathway throughout the cell cycle including S and G2, the highest HR rate was measured in S-phase (but still much less than NHEJ). I would be interested to lean about similar studies in yeast or plants.

Thanks a lot!

Gertrud

Is anyone experienced with assays to distinguish non-homologous end joining from homologous recombination in DNA repair?

Hi,

I ran the CellProfiler using the ExampleFluorescentCometAssay.cppipe pipeline. However the output .csv file for comet tail do not show %tailDNA for the analysed comets. Is there a module that can be added in the pipeline to calculate it? How can I use data like AreaShape_MajorAxisLength, AreaShape_MaxFeretDiameter or mean intensity corrgray to translate into %tail DNA or DNA damage.

Thanks

The genotoxic effects of UV are well known, which includes the formation of pyrimidine dimers,  several photoproducts, and the strand breaks (single and/or double strand). Most of these defects are repaired within 48 hours of the radiation exposure through ‘DNA repair mechanism’ and no traces of these would be expected in a long-term unless the damage is severe enough to be recovered by repair mechanism, which in turn would lead to mutagenesis.

I am planning some experiments to investigate the Genotoxicity of UV exposure on an animal cornea. What DNA defects that can be identified in the laboratory would I expect after the fourth week of radiation exposure (as most of them would repair)? Clinically cornea would show its stress in the form of opacities and pigmentation (? and probably oedema as well).

I am performing ChIP on a DNA repair factor in budding yeast. We are interested in looking at the enrichment of this factor in WT and some mutant strains. And I am using a strain that can induce a single locus-specific DNA double strand break upon shifting the carbon source to galactose. I have my DNA repair factor tagged with 3xFLAG and I'm using EZ View preconjugated FLAG beads for my IP. 

I collect cells before Galactose induction and at various time points after galactose induction, use IgG as mock IP and primers at various distances from the DSB sites to probe for enrichment.

So far, I have only performed ChIP in my WT strain and I'm getting some weird results. When I normalize my qPCR results, I usually get the highest enrichment from non-induced sample and the rest of the samples show some enrichment but are much lower than my non-induced (no DSB) which is odd to me as I expect higher enrichment when DSB is induced. 

I am not sure if the problem is with more cell lysate in later time points (as cells still continue to  grow for some time after DSB induction). Since what I do after lysis and sonication, is to save 1% as input and the rest of the lysate is divided equally between mock IP and FLAG IP for all time points (regardless of how much cells I actually collected). Does anybody have any recommendations on how to troubleshoot?

Are there other general DNA Repair markers (phospho-proteins) besides gamma H2AX (S139) suitable for flow cytometry analysis?

.

Why familial mutation in RB gene prefer to give rise to Retinoblastoma, why not other type of cancer ?

(or)

Why familial BRCA gene prefer to give rise to Breast cancer, why not other type of cancer?

RB gene participate in cell cycle regulation  which is not unique to retinal cell ,it participate in all types of cell, then why familial RB gene mutation prefer to give rise to Retinoblastoma (Eye cancer). Similarly BRCA gene participate in DNA repair mechanism in all types of cell, not only mammary tissue . then why do familial mutation in BRCA prefer to give rise to breast cancer ,why not other cancer.

I am trying to modify a mutation assay I have had success with in other suspension cell lines - PIG-A and have encountered some difficulties that I am hoping to troubleshoot with you all. PIG-A is a transmembrane (GPI) anchor, which functions to tether a variety of cell surface molecules to the cell surface. A null mutation in the PIGA gene results in the loss of this transmembrane anchor protein, and also the loss of all of these tethered cell surface receptors. Hence PIGA mutants can be detected by flow cytometry by looking for the absence of two or more of these cell surface receptors (to ensure that mutations in the genes of the cell surface receptors alone are not counted). I have had previous success with this assay in a lymphoblastoid cell line and generated some excellent mutation data which is about to be published. I am trying to adapt the assay for a myeloid cell line (HL-60), but am encountering problems.

The two GPI-anchored cell surface receptors I am looking for are CD55 and CD59, both of which are highly expressed on HL-60 and show significant binding with fluorochrome-conjugated antibodies.

As well as this, I am concurrently using an antibody to bind CD45 as a positive control and 7-AAD to exclude dead cells.

All antibodies show excellent binding, but even in 500,000 cells, I am not picking up a single one which appears to be a PIGA mutant (CD55 and CD59 negative). I have pre-treated the cells with an Fc Blocking agent to prevent non-specific binding of the antibodies and have checked the isotype controls to make sure there is no non-specific binding. I have also treated cells with radiation at a dose which I know should cause significant mutation as a positive control - and cannot see any PIGA mutations here either (8 days post treatment, which initially caused around 70% cell death). I would absolutely expect to see at least 5-10 PIGA mutant cells in 500,000 events, and far more in the radiation treated cells.

A few things I have considered:

I am using too much antibody and it is nonspecifically binding to everything causing false positives (I shall titrate it this afternoon and try with less antibody).

My compensation for the assay is incorrect and I am not seeing negative cells due to leakage from other channels (NB: All cells I use in the assay are EGFP+)

PIGA mutation is fatal in HL-60 but not in the previous lymphoblastoid cell line I have used.

HL-60 have a very low basal PIGA mutation rate and I have not left it long enough after the radiation treatment to allow the cells to fix the PIGA mutations and lose cell surface expression of CD55 and CD59.

Please note in attachment (gating strategy for PIGA) - CD19 is the control marker used in the lymphoblastoid cell line and is now CD45 in HL-60.

I realise that this is a quick summary of a not incredibly simple assay, which many will be unfamiliar with, so please do ask for more information/clarification if you think you might be able to help, i'll try to respond quickly.

Many thanks for any help - I'm at my wits end with this assay - there is no theoretical reason why it shouldn't work, but I am definitely not picking up any mutants at all.

Additionally - if anyone is interested in the assay - there has been a publication about it here (with different cell types to that I am using) http://www.ncbi.nlm.nih.gov/pubmed/20034593

What is the shipping condition of Bio-Rad iScript™ cDNA Synthesis Kit?

Is it know whether nucleated red blood cells in reptiles, birds, or fish can undergo DNA repair? I would have assumed that someone has looked into this at least in chickens, but I have not been able to find any articles on this.

Dear colleagues! I very interested in deep understanding of DNA DSB reparation after preoperative radiotherapy. It is essential for me to understand exact mechanisms of B-NHEJ and D-NHEJ reparation. We need to assess therapeutic response and try to use reparation systems activity as a marker. Unfortunately, I find insufficient information in literature. Please help me if you are interested in this field too.  

In our data I have found that down-regulation of cell cycle genes is accompanied by down-regulation of DNA repair and replication genes. So - I'm wondering if there are any other reasons for cell cycle arrest other than DNA repair? 

want to know from different peoples

Does someone knows a good book or a PDF file on the subject of DNA repair? Especially with BRCA1 as a key gene? It is for a thesis defence. So it shouldn't be too extensive. 

Hey guys, I am a fresher in DNA damage field and am trying to detect NHEJ and HR effects after target gene knockdown. So, can somebody give me suggestions about how to detect NHEJ or HR, like detect some genes involved or see some genes' immunofluorescence, or show me some helpful papers in which there are some ways to detect these.

Thanks!

Since homologous directed repair has very low probability to occur in bacteria, can we somehow increase the probability by arresting cell division and inducing SOS response in bacteria in exposure to damaging agent in low concentration? What kind of substance can we use to induce SOS response? thank you.

What are the subtypes of genome instability and the genome instability caused by the misrepair of DNA double-strand break in proliferating and nonproliferating cells,respectively?

Hello.

I'm using CRISPR technology for the first time. The kit I'm using is GeneArt® CRISPR Nuclease Vector with OFP Reporter Kit (Catalog number: A21174, Invitrogen).

My question is how much time should I wait from transfection (by Lipofectamine 2000) until being safe to use FACS for OFP enrichment of my cells (HEK).

Also, if I want to introduce puromycin (~600bp) resistance after the DS break, what kind of plasmid should I use for the homologous recombination repair?

Thank you!

I am planing analyze yeast deletion strains having abnormal telomere length. I want to know about best methods for telomere length assay and also about commercial available kits. how to synthesize yeast specific telomere probe labelled with DIG?

Hello,

Does anyone know of any DNA/RNA repair proteins that can be used as a marker to detect localised damage within the nucleoli of cells - such as exposure to radiation?

Nucleoli are made up of mostly RNA so if they received exposure to radiation producing single stranded breaks, would these be repaired by DNA repair proteins or RNA proteins?

Thank you for any help or guidance

These days I'm doing DNA repair experiment, first use restriction enzyme to cleave the DNA, then two enzymes, then use ligase to religate, but the bands (repaired) always blurred, and in different size (larger a little bit). I didn't purify the product, so the reason, reaction buffer contains high salt? or PH influence the shift? thank you all very much.

In every illustration and available resources it says that the two major pathways for repair of DSB is either homologous recombination (HR) or nonhomologous end joining (NHEJ) in crispr cas9 system 

how does this possible in artificially introduced systems (e.g in plants)? Do they inherit required enzymes / proteins naturally for this kind of situation?

As I've understood if I introduced gRNA to plant for crispr cas9 system, it will create the DSB, if however this break repair by HR, again DSB will create by the expressing gRNA  crispr cas9 system....Unless if there will be a insertion / deletion / mutation this process will repeat  like a cycle?? 

Also, suppose that we introduce Homologous repair template along with the Cas9-sgRNA , So is this mechanism that much effective and efficiency of screening requires condition of our experiment in plants.

I wonder what actually happen actually? Can some one explain this :)

I would Like know whether it is possible to study the proteins and there concern genes involved in the DNA break repair and homologue recombination using a Real time PCR with out employing transfection studies. 

I read about DNA repair after exposure to UV light, photoreactivity is one of the mechanisms of repair. This mechanism is performed in the presence of light. I am wondering, if somebody exposure the spores suspension or vegetative growth (fungi grow on medium) to UV light how long need to left the plate or spores suspension in room to perform DNA repair?  

Regards

I am trying to detect CPD foci in cells following UV-irradiation via Immunofluorescence Microscopy. What is the difference, if any, in the intensity and the number of foci formed when the cells are treated with UV, globally as opposed to locally (using a micropore filter)?

I am currently looking for a near diploid human cancer cell line, such as HCT116 and U87-MG. Preferably XY with modal chromosome # between 40-50, "normal" DNA repair pathways, and able to easily be transfected and cloned. Does anyone have any suggestions?

The basic GFP+ signals in my sistem are very low (about 1%) because the majority of DR-U2OS or EJ5 cells undergo apoptosis when transfected with I-sce1 plasmids.

I mean: after what time from start exposure, DNA damage is started and after what time, repair is started?

if possible, attaching a file for this method please.

It has been demonstrated that double-strand DNA break response proteins, such as TP53BP1 and RIF1, are required for NHEJ; DNA double-strand break repair pathways proteins such as KU70/KU80,DNA-PKcs,XRCC4 and so on, are responsible for repair. However, I could't find papers about how TP53BP1 and RIF1 regulate repair pathways? If you have read same paper, please share with me. Thanks a lot

I am studying DNA binding of a protein using EMSA. The reaction mixture contains 5 nM of DNA substrate while I vary the protein concentration from 2 to 50 nM. I see an increase in band shift with progressive increase in the protein concentration ultimately reaching binding saturation at 50 nM protein. However, it does not lead to 100 percent binding. What might be the reason behind the binding curve not reaching 100%?

in fact I want using gill and liver cells.

Many intracellular processes, such as DNA replication, repair, cell cycle transitions, nucleus and spindle assembly etc., can be recapitulated and studied in a cell-free setting, e.g. in Xenopus egg extracts. Does anyone know whether oxygen concentration or light spectrum have an effect on any of the above or other processes modeled in cell-free systems?

Thank you.

We and others have found a low activity of DNA damage repair in subjects with cancer compared to healthy subjects. However, we don't know if altered activity repair is a cause or a consequence in cancer development.

Is there a specific antibody for western blot or immunofluorescence?

We all know that cancer is mostly caused by rare genetic mutations of a specific gene. We also know that DNA polymerase III repairs mismatches of base pairs during DNA replication or recombination processes.

As DNA polymerase III is always responsible for this proofreading or correction, why does DNA olymerase III can’t repair the base pair mismatches that cause cancer?

I am looking into inducing p53 specific activity in thymocytes but can only find papers where they induce DNA damage with y-irradiation. Any direction would be much appreciated.

I am using gel bond film for the comet assay rather than agarose coated slides, but am struggling when it comes to the elctropheresis as I can't be certain that the film remains straight and isn't moving around in the tank.

Seems like there are lots of resources about the nuances of generating gRNAs, but a little less on designing HDR constructs. Just a few quick questions:

1. If my gRNA is ~50 bp away from where my point mutation is, do i want to place my arms of homology around the point mutation or around the cut-site? Or would I be better off putting 50bp upstream of my point mutation and 50 bp downstream of the cut site (if cut site is downstream of point mutation)

2. Can my cut site lie outside the arms of homology by a few bp, or is this not effective (donor DNA construct must flank gRNA site)? If I can do this, won't Cas9 just re-cut at that location --> NHEJ --> indel, or am I misunderstanding this as a potential risk?

Thanks!

I am over-expressing a protein of interest (implicated in DNA repair) in fusion with yfp in Nicotiana Benthamiana leaves. The proteomics data showed that the protein is cytoplasmic as well as chromatin assotiated. The microscopic studies done at the same time showed high cytoplasmic expression for the protein but couldn't detect the protein in the nucleus. Under YFP filter, I can see fluorescence around the nucleus and faint color inside. I used as a control for nuclear localisation mcherry "expressed by the same construct" which showed very bright fluorescence. I am just wondering if there is a technical issue that hinder the bright nuclear fluorescene (could be the high cytoplasmic fluorescence!) and how can I optimize the experiment to be able to see the nuclear fluorescence. Thank you.

Telomere FISH revealed that our primer human cells have de novo telomere at the broken ends of the chromomeres arms (mainly at the centromeric or pericentromeric region of the chromosomes as the breaks are at this part of the chromosomes). So, I would like to gather information about this kind of repair mechanism.

Telomere FISH revealed that our primer human cells have de novo telomere at the broken ends of the chromomeres arms (mainly at the centromeric and pericentromeric region of the chromosomes as the breaks are at this part of the chromosomes). I would like gather information this kind of repair mechanism.

For the quantification by densitometry of supercoiled and relaxed forms of plasmid DNA stained with ethidium bromide and separated in agarose gel there is a correction factor of 1.4 because the relaxed form gives a fluorescence intensity 1.4-fold higher than the supercoiled form. Should the same factor be used to perform the quantification of plasmid DNA stained with GelRed?

We know that HL-60, Jurkat and MOLT-4 are p53- or mutated. What about MonoMac-6 and MOLT-3? We are involved in the evaluation of DNA repair (adaptive response)

The question is regarding what determines the output (high or low) of DNAPK activity detected using the Promega SignaTect DNAPK assay kit. The assay starts with same amount of nuclear extract, with endogenous DNA removed. DNAPK will then be activated by the addition of dsDNA and starts phosphorylating a p53 fragment, and DNAPK activity is measured by the incorporation of radioactive ATP. Question is, does this mean that as long as there are equal amount of Ku and/or DNAPKc in the nuclear extract, the DNAPK activity measured by this assay will be the same? In other words, this assay simply reflects how efficient is DNAPK translocated to the nuclei? Does existing DNAPK activation/autophosphorylation affect the output of this assay? For example, if the experiment and control samples have a SAME protein level of DNAPK in their nuclear extracts, but experiment sample has already activated DNAPK (likely retains the autophosphorylations T2609, S2056), will the experiment sample have higher DNAPK activity even though DNAPK protein levels are the same?

Role of hydroxymethylation.

I was reading a paper and they incubated fibroblast cells in caffine (1mM) for an hour after UV irradiation. I was wondering what was the reasoning behind this?

We are working on the relationship between DNA damage and ageing.

I am preparing slides for the comet assay. I have dipped my slides into normal melt agarose that has been pre-warmed. However it is all evaporating off my slides after about 15 minutes. Any suggestions much appreciated!