DNA Replication - Science topic

Phil Geis,

As for your suggestion "...execute this "simple" synthesis of life and show us", I wrote in the previous post the following: "I know no such examples in the history of science, when the theories of so large scale and the results of their practical realization were presented by the same scientific teams".

@Robert Adolf Brinzer The question is, what is the possible structural and functional role of newly discovered SIRC (Short Interrupted Repeat Cassettes) elements in plant genome. The point is that in bacterial genomes there are CRISPR cassettes which are looking similar to SIRC but have nothing common.

In certain DNA replication errors, particularly during template-switching events like strand slippage or misalignment, the resulting duplication can indeed be either in the same orientation (tandem duplication) or inverted (inverted duplication) relative to the original sequence. This can occur under specific circumstances and mechanisms during DNA replication.

In template-switching, which is often associated with replication errors like slipped-strand mispairing, the DNA polymerase temporarily dissociates from the template strand and switches to a nearby template strand before continuing replication. The orientation of the newly synthesised DNA strand and the template strand to which it switches can determine whether the resulting duplication is in the same or inverted orientation. Here's a basic explanation:

The specific circumstances and outcomes of template-switching events can vary, and they are influenced by factors such as the length and sequence of the repetitive elements in the DNA, the timing of the switch, and the precise mechanisms involved in DNA replication. These events can lead to genetic rearrangements and contribute to the diversity of genomic structures, which can have implications in terms of genetic variation and evolution.

Ryan Gourlie

The pattern changes during replication as different parts of the genome replicate at different times during S-phase.

Bhawna G. it's purposefully created to be non-amplifiable for you always have to purchase it from the company. It's just for the company makes more money.

There is some evidence to suggest that plastic pollution may cause DNA damage and mutations in aquatic organisms. Plastic particles can act as carriers for other pollutants, such as polycyclic aromatic hydrocarbons (PAHs) and heavy metals, which can be harmful to DNA. Additionally, plastic particles can generate reactive oxygen species (ROS) when they come into contact with UV radiation, which can also damage DNA. A study published in 2021 found that exposure to microplastics caused DNA damage and altered gene expression in the liver of zebrafish. However, more research is needed to fully understand the effects of plastic pollution on DNA damage and mutation in aquatic organisms.

Hi,

I'm bringing up this post!

Sanjay Premi your sequencing results gave me some hope ... Let me explain, I try to amplify yan ITS target on grapevine DNA, expected amplicon size : variable around 200 and some.

Here is what I get

Without DMSO: non reproducible results between samples or between pcr on the same sample. In general: very weak amplification (bands not visible or very weak), one or more bands, very variable band sizes, some are > 500 bp.

With DMSO: VERY reproducible results between samples and PCR, ALWAYS only one very visible band slightly < 500 bp.

I have not done any sequencing for the moment.

Sanjay Premi if you still have pictures of your gels with and without DMSO, are you ok to share them with me? or do you simply know more about the origin of this?

Kind regards

Paola

First you should specify if your are interested in a particular gene, its gene expression, or just genomic DNA. If you are interested in a gene or a specific genomic or plasmid region, then qPCR is a good method. Just design primers for that, and for a control gene or region, and build a standard curve with known concentrations or copy numbers to extrapolate fluorescence intensity values

Dear Clarence Hu

EBV seems to employ epigenetic mechanisms to control the host transcriptome and to control expression of its own virally encoded genes. Upon initial infection of a cell, the unmethylated viral genome can undergo viral replication with new virion production, while a subset of infected cells acquires a highly methylated viral genome that squelches expression of foreign proteins and mediates long-term viral persistence by way of latent infection. Infected tumors tend to have highly methylated EBV DNA, and methylation-related silencing of viral genes helps explain how infected tumors evade immune destruction.

The reagents mentioned by you like 5-aza-deoxycytidine (5-aza), Sodium butyrate, Tetracycline derivative doxycycline (Dox), etc. are demethylating agents with the capacity to induce transient DNA hypomethylation.

Deliberate attempt to reduce the DNA methylation in EBV-associated cancer with the aim to activate the expression of cellular and viral genes has the potential to achieve two potentially therapeutic events.

First, it may result in reactivation of the expression of viral genes that expose the cells to attack by the immune system (the recognition of the target by virus-specific cytotoxic T-cells).

Secondly, it may result in the expression of important cellular genes that cause the cancer cells to stop proliferating or to die through apoptosis.

So, deliberate attempt to alter DNA methylation presents an attractive research direction which may lead to new therapeutic approaches to treat EBV-associated cancer.

In a way, yes, it is specific to EBV because EBV employs epigenetic mechanisms namely, attains a highly methylated viral genome that helps mediate long-term viral persistence by way of latent infection evading the immune system. When demethylating agents are used, they reactivate viral gene expression and as a result are easily recognized and attacked by the immune system.

I have attached a research article for your reference.

Best Wishes.

There are x-ray structures of DNA molecules that are not oligo's (although also many are oligos). In fact the Watson and Crick model is based on that and numerous others since then.

There are undoubtedly many forms of DNA, (A-form, B-form, Z-form), triplexes etc. which are found in the laboratory and in nature, so I would never say your model is not possible under some circumstances, but the experimental evidence for B-form being the norm is overwhelming.

I agree with Malcolm Nobre

Well-known examples are aminoacyl-tRNA synthetases and acyl-AMP synthetases, which generate pyrophosphate. The subsequent hydrolysis of pyrophosphate by pyrophosphatase, which requires Mg ions, is an exergonic reaction that further drives the first reaction

The first mechanistic model of self-replication was given by John von Neumann by his self-replicating cellular automaton. He was followed by others: Langdon, Reggia, ...

It would be interesting to study this research stream as it provides great insights into creation of mechanistic description of certain properties of living structures.

Hi , try using the reverse sequence results

The SAC comprises two arms: tension and kinetochore occupancy by microtubules. If SAC is active (and in some early embryos/oocytes it is not: see Castagnetti et al Cells, 2020). One other important aspect of the SAC in oocytes (mammalian) is that it regulates the normal duration of meiosis I from GVBD to Ana I (see Homer et al., Genes Dev. 2005). However, for human trisomy it is possible that Rec 8 protein levels fall with maternal age, weakening cohesion between homologues which because of a non-effective SAC leads to aneuploidy.

Great points. Thanks

If I'm not wrong the the replication origin contain all the sequences necessary for the assembly of the machinery that is compose by several subunits.

You've basically answered your own question. The DNA polymerase won't allow an RNA nucleotide as an INCOMING base. But it only needs a 3' -OH group to start polymerizing more DNA.

@_Subhash C. Juneja, as per our knowledge methylation leads to silencing , and in case of replication it contrasts , as it is needed for polymerization to get start

Thanks for your answer. I am trying with U2OS cell line using 3J/cm2 in total. My cross linker has a program where I put the amount of J/cm2 and the machine will give to my cells. It takes 10 minutes to dispense the total dose of radiation. What I saw using EdU incorporation and DAPI assay after 12-24-72h, is that, cells strongly accumulates in G2. Also I found online many papers where they title the UVA dose in response to cell viability. The majority of that papers use a dose between 0.3 J/cm2 and 1J.

Check: PCR Protocols - A Guide to Methods and Application; edited by, Michael A. Innis, David H. Gelfand, John J.Sninsky, Thomas J. White. Published by Academic Press.

Thank you all.

I also find a very good review on the topic:

Genes (Basel). 2017 Feb 8;8(2). pii: E62. doi: 10.3390/genes8020062.

Elaborated Action of the Human Primosome.

Baranovskiy AG1, Tahirov TH2.

I think this is quite a difficult question to answer. DNA replication has far more components and scope than MMR which is only a subset of all DNA repair. That being said MMR does have a strong link to carcinogenesis. Much of this decision hinges upon what your plans are when you finish your thesis, do you plan on going into academia or industry? I had a similar choice when i was beginning my scientific career and picked DNA repair. I have been happy with my decision, partly because there is a huge amount of disease relevance (think grants) but also because it gave me avenues into other related fields such as genome engineering.

Thank you very much!

I'm still interested in additional opinions/theories. So, if there is anyone who is willing to contribute to this topic please don't hesitate to drop a comment. Thank you!

Dear Dr. Sukayna Jabbar,

Sorry, I don't understand you. Please, express your thought in more detail.

Hello, Non-homologous end joining is a pathway that repairs double-strand breaks in DNA which is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair, which requires a homologous sequence to guide repair.

i agree with Victor Ostrovskii

Have you interest in differences in molecules employed? In Processes of detection? Please, give a bit more detail to your question.

Hi Aydin,

You can observe this phenomena mainly in retroviruses and DNA viruses that require integration of genomic material into the host cell. The primary reason is the transcription of viral proteins solely depends on the host machinery. Hence, establishment of an infection is more critical than viral replication. Thus, if we inhibits viral replication at the early phase of an infection we can contain the infection. That's why we provide drugs to inhibit viral entry and viral replication. The activation signals that required for expression of late genes is often linked with a set of operon(s).

Theoretically, expression of operon itself contained thereby expression of late genes also contained. There is a threshold for release of viral progeny to infect new cells. The proteins encoded by late genes were often related with release of viral progeny not for establishment of an infecion (at early phase). Hence, inhibition of viral replication certainly switch-off the expression of late genes until the threshold for release of viral progeny or newer virus to infect further or bystander cells. I understand that the inhibition of late gene expression (via natural process) provide sufficient time to establish infection via evasion of host immune response. The remnants of proteins expressed by late gene also enhances the probability for detection of host immune machinery. I think the fine balance between the survival strategy of the virus and host leads to evolution of late gene expression under stringent regulation

Dear Kim

The reversibility of the enzyme inactivation at high temperature was tested show that sometimes a re-versible inactivation at higher temperatures is observed.

Thanks all

Hello Vipin

Have you looked at the cell cycle profile after the depletion? The genes that are down-regulated after the depletion of your protein of interest, are they regulated in a cell cycle manner or are they constitutively expressed? Is it possible that depletion of the replication factor is causing a cell cycle arrest that is the reason for the down regulation of the other centrosomal factors? You could also look at the RNA levels (of the centrosomal factors) after over expressing your candidate replication factor. Have tried to add back or express the replication factor (of interest) after RNAi to check if it can reverse the effects observed after RNAi depletion.

You can email me if you want to discuss more.

Dear, how you can activate oocyte with progesterone

@ Frieda It is possible to alter genes but I am opposed to changing what comes from with-in from past to future; it involves very large time periods and acquired balances that are very complex. I think we should always care for the sufferring; drugs, etc. applied to symptoms, to alleviate suffering are OK.

Concerning intelligence genes might only have a role in having the ability to think and no role in intelligence or what is thought. People think and relate in the present according to life experiences and situations from where they gather their knowledge. 

Reference:

I think the metabolic environment changes and the environment changes.

In short periods of time, it has to do with the metabolic environment. Changes in the length of time (long lag time) are associated with changes in living conditions.

The key is the position of the so-called normal environment.

Hi Nikolaos,

Did you find any good results for your POLQ western Blot?

Thanks for your thorough explanation that cleared up the subject to me. You are absolutely right, the newly synthesized DNA won't dissociate from parent strand and it will be purified along with rest of DNA. I had a wrong assumption that I may loose these newly synthesized strands if purification method excludes small pieces of DNA> Thanks a lot again for your help Ali, I appreciate it a lot. 

WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations

BMC Genomics20078:275

DOI: 10.1186/1471-2164-8-275

I second Inger. Store the cells in PBS after fixation, but not in the permeabilisation buffer!

Dear Tafrihi,

A very intriguing question! DNAP can only catalyse the SN2 nucelophilic attack of 3' OH to the alpha phosphate of the incoming complementary nucleotide. So it needs a primer-template junction to begin adding the dNTPs to the new strand. But, it cannot add the first nucleotide where it has to hold on to the cognate dNTP and then add the next nucleotide to it. Whereas, RNAP takes the first NTP (which remains as NTP as there is no nucleophilic attack on it) holds on to it and then adds the 2nd and subsequently all the nucleotides to it. This difference makes DNAP a very specific enzyme which can only polymerize or extend an existing chain. This probably gives DNAP an evolutionary advantage, as most misincorporation of nucleotides tend to occur in the initial stage where the base pairing between the new strand and template strand is less, so if there is a RNA primer initially, which is of sufficient length, then there is lesser chances of misincorporation. Later the primers are replaced by DNAP I. But for RNA synthesis, that high fidelity isn't required, so RNAP is allowed to start de novo. But any such errors by DNAP at the initial stage will slow down the replication progression, which is avoided by the primers incorporated by primase and later carefully replaced by DNAP. I think this could be one the reasons why DNAP can't initiate de novo synthesis while RNAP can.

There is a publication in which cytoplasmic ROS levels in the filamentous ascomycete Podospora anserina (which is closely related to N. crassa) are studied by using the HyPer protein (please see attachment). You could think about constructing a strain that expresses a gene encoding the HyPer protein with a mitochondrial targeting signal. There is no guarantee that it would work, though. When I was working with Podospora anserina I was not able to find a fluorescent dye that reliably reports on mitochondrial ROS levels (usually the background levels were much too high). But you could consider testing MitoSOX which seems to be a good probe for measuring mitochondrial oxidative stress in mammalian cells (I didn't test this dye for use with Podospora anserina so I can't give you any hints).

Regards, Christian  

Your protocol suffers from different problems:

The short incubation time which may be one of the most important problems has been cured. We use proliferation tests in a clinical setting and allow routinely 16-20h of BrdU incorporation.

The next important step is to remove ALL unbound BrdU from your cells as most antibodies do not differentiate between BrdU incorporated in DNA or being unboing in the well. Wash your cells several times with PBS with 0.05% of TWEEN. You have to centrifuge your plates between every washing step to avoid cell loss. Do not tap the plate on paper towels etc. as this will cause cell loss! We do not use a kit, so our last step is to fix the cells with ice-cold ethanol. Again, the plate is centrifuged to have all cells at the bottom of the well. This may be important for proper BrdU detection by the antibody.

After drying the plate, denaturation is an important step. You may test either HCL (1-2N) or microwave, both work well in our hands. If you do not see any colour development adding your chromogen do not stop the reaction but wash your plate and start again from the denaturation step.

It is also highly recommended to add a control without BrdU to get the background values of your antibody. Subtraction of this background from the whole plate values is important in the plate analysis.

There are several forms of PHA which have varying capabilities to stimulate lymphocytes. Overdoses of PHA will kill the cells. Using PHA-L which is the most potent stimulant the concentration should not exceed 5µg/ml. Optimal doses of PHA-P or PHA-M have to be estimated as they are crude preparations with varying stimulatory capacities. Anyway, 50µg/ml seems to me as a very high dose.

I hope that these suggestions may help to solve your problems.

regards

Gernot

you can try anti-Brdu antibody (BD biosciences CAT No:347583).

The Barkai lab answered my question in a paper in Science this week!

Expression homeostasis during DNA replication. (http://science.sciencemag.org/content/351/6277/1087.full)

Doubling DNA but not expression.

As the genome replicates, and before the cell divides, the copy number of the replicated portions of the genome doubles. In bacteria and archaea, gene expression tracks with gene dosage, both of which increase after DNA replication. Voichek et al., however, show that an increase in DNA dosage after replication does not increase gene expression in budding yeast. This expression buffering is mediated by the acetylation of newly synthesized histone H3 deposited on the replicated DNA. This acetylation helps suppress transcription from the excess DNA.

The US NCI has tested (and continues to actively test) not far from one million compounds.

They analyzed the drug-induced effects on cancer cell population growth after 48h.

Sharing at 100% the remark of Paul, we always use 72h of cell-drug incubation.

It is obvious that data generated after 24 or 48 or 72 or 96 hours will totally differ from each other for a same compound on a same cancer cell line and with a same concentration.

However, unfortunately, cancers in patients are very heterogeneous and their cell doubling times amazingly changes from one subclone to another one in the same cancer.

We have to keep in mind that some "poor cancer cells prinsoners of plastic flasks" are far to be representative of the "biological reality" in a cancer developing in a patient.

Thus, I think that it is better to "stay" on 48 or 72h to be able to compare "our" data to those generated by our colleagues.

I am attaching here articles relating to the NCI 60-cell-line approach and also some articles on the complex biology in "true" cancers.

Best regards

Robert

Dear Ismali,

I think you read something in other context and interpreted as 3 mutations limit. I am

sorry there is something wrong.

Hello Anna. Basically you are looking for shuttle vectors that can be conjugatively transferred from E. Coli to your organism of interest. Whic replicon they should contain for autonomous replication in host cell is something you got to find out in the literature.

Both required enzymes and primers

Hi Parham,

If I understand your question correctly, you are asking what is the shortest time between addition of the analogue to medium and incorporation into DNA assuming, of course, DNA synthesis is going on. I have no experience with yeast, but in mammalian cells it takes very few minutes at the most. You can pulse-label the cells for 15 minutes (from addition to medium to fixation for staining) and still see a very clear signal. Also, still in mammalian cells, you can use two labels one after another with a simple wash in between and there is no overlap and no gap between the two labels (used for DNA combing). Thus, in mammalian cells, uptake and washout are very fast. I'd assume it's essentially the same in yeast.

I hope this helps

Are there any reports/ observations that pET22b  can arrest DNA replication in E. coli? If that is the case, you cannot transform any E. coli with this plasmid.

pET22B is similar to otehr pET plasmids. It is specifically used for secreting proteins to periplasmic (pelB signal sequence), for more control during protein expression (lacI gene) and for tag based  detection and purification. I don't think it has any role in DNA replication in E coli

This paper may help - gives quite an in depth analysis of EdU compared with older BrdU examples

we are using it only for replication fork and origin studies. I think that the bottle neck in this procedure  is to obtain long and intact dna molecules.

I think you have the cell cycle mixed up.  During S the nuclear membrane is intact, it does not break down until mitosis.  The areas around the replication forks during S is where the DNA is uncoiled.  Also anywhere there is active transcription at any point in the cell cycle.

I think the answer of  Masashi is true

Hi Ariel,

this is an interesting question. However I don't think, it can be answered generally, because here you need to specify "RNA synthesis" more precisely. For example, the cell cycle-specific gene expression will definitively be affected by any perturbations in the progression of the cell cycle. In other words, the G2/M genes or histone genes wouldn't be expressed in thymidine-arrested cells, unless you release them from the arrest.

On the other hand, the basal transcription is probably not affected by thymidine, because if the G2 cells are treated with thymidine (for example after first release in the double thymidine block), then the cells are still able to progress into the new cell cycle and only arrest at the beginning of the consecutive S phase, that, in my opinion, assumes they are transcription-competent.

I have also two additional comments regarding your purpose.

1. If I got it right, you are working with the ES cells? The duration of the cell cycle in the ES cells is considerably shorter than in somatic cells (10-12h vs. 20-24h), so may be you can also shorten thymidine treatment to avoid prolonged cell cycle arrest and so minimize apoptotic cells.

2. Thymidine causes S phase arrest, regardless of its progression, so if you just do a single thymidine block, you then get a mixture of S phase cells (early, mid and late). This might be sufficient sometimes, but if you want to monitor the progression through the S phase and to compare early vs. late S phase, then the double thymidine block is a better synchronisation method.

Good luck!

Jena Biosciences has Atto647N labelled bases. This dye is equivalent and better than Cy5.

سلام آقای دکتر

ممنون، آره اون مقاله شما رو مشاهده کردم. بسیار عالی.

من ابتدا شک داشتم به اشتباه در پی سی آر و جند بار با روش های مختلف استخراج کردم دی ان ای رو و دو بار سکانس کردم. تا مطمن شم. اما یکم هنوز باور اینکه حذف 50 نوکلوتاید به طور همزمان {احتمالا قابلیت پروف ریدینگ در جهت عکس عمل کرده} در حداقل صد نسخه دی ان ای ریبوزمومی در یک قارچ و وبقای اون یکم عجیب به نظر میرسه.

The replication origin is unlikely to be at the very end of the chromosome. Therefore, the end of the leading strand is most likely to be covered, which leaves the lagging strand to be the only problem, ideally. 

Hello.

I am no expert, but you may want to read this paper.

Srivatsan A, Tehranchi A, MacAlpine DM, Wang JD. 2010. Co-orientation of replication and transcription preserves genome integrity. PLoS Genet. 6:e1000810

Best,

Lim

Thanks, then could  be a point mutation that leads to no change in the nucleotide sequence? Consider a double-stranded sequence:

(5') GAC

(3') CTG

and a mutation happens on the 3' as (3') CAG

then after the DNA replication the sequence becomes:

(5')GTC

(3')CAG

This is the same as the original sequence?

Jing

Andrey: Are you saying that sections of DNA are removed from differentiated cells; that they do not retain the full complement of DNA?

In principle, oxygen concentration or light spectrum is a important factor of DNA repair, replication, as well as recombination in living system. Thus, the question invokes whether molecular physiology is relevant for a model system in terms of variations measurement. Considering a cell-fee system is a model of life soup per se, the robustness of interested measurement is largely context-dependent. It appears to be loss of compartmentalization for cell free system thereby such a distorted organization leading to a distinct chaotic signaling system which is unknown. Thus, an intricate system is required to model oxygen concentration or light spectrum as a factor especially considering the reaction speed and the time to reach equilibrium.  Together, it might be crucial to employ an oxygen concentration or light spectrum sensitive enzyme to examine all these speculations. I was wondering which protein is ideally suitable.

It depends what you want to see. Carmine acetic acid works. You could also use other methods  like Giemsa staining of fluorecence methods dependent on your question. Usually simple phase contrast will be nice if you understand to make suitable slides.

creates specific probes, your question is not clear or precise thank you

Think about PCR

DNA polymerase III is prokaryotic. We eukaryotes have several different DNA polymerases, some having proofreading activity, some being less careful but can effectively replicate through difficult structures such as DNA lesions or stalled replication forks (translesion pols), at the expense of fidelity.

Not all replication pausing sites are converted into breaks. Here is a nice review where it is discussed in detail: Labib, K., and Hodgson, B., (2007). Replication fork barriers: pausing for a break or stalling for time? EMBO Reports 8(4):346-53..

One theory is that the Dnmt1 isoforms are present, but they travel from nucleus to cytoplasm (and back). Dnmt1 can only be active in the nucleus as there is no DNA to be methylated in the cytoplasm. These are some of the first and most important publications on this topic:

Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development.

Ratnam S1, Mertineit C, Ding F, Howell CY, Clarke HJ, Bestor TH, Chaillet JR, Trasler JM.

Regulation of stage-specific nuclear translocation of Dnmt1o during preimplantation mouse development.

Doherty AS1, Bartolomei MS, Schultz RM.

----

This publication is more recent and analyses oocytes (also Dnmt1 knock-outs if I remember correctly) using more up-to-date methods:

Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases.

Shirane K1, Toh H, Kobayashi H, Miura F, Chiba H, Ito T, Kono T, Sasaki H.

@IIya,

Probably Abbas meant to say 'before DNA sequencing technology' was all over the places like today.......

Often plasmids used in yeasts (S. cerevisiae) are so called shuttle vectors that you can use in bacteria or yeast. Using different markers (LEU2, URA3, HIS3, etc.) you can transform yeasts with several different plasmids (best one after the other).

Next to a marker, these plasmids contain an "ARS" (autonomously replicating sequence) and often a "CEN" (yeast centromere); they replicate like chromosomes and their copy number is regulated (on average 1 plasmid per yeast cell).

You can also use derivatives of yeast 2 µm plasmids, which lead to higher plasmid per cell number or even integrative vectors to insert your protein-of-interest genomically.

For high amount protein expression, I can highly recommend the yeast Pichia pastoris. This methanogenic yeast can yield a very high protein expression (protein-of-interest is genomically integrated and under Aox1 promoter; using methanol as C-source can result in up to 30% of overall protein in the cell).

I hope, I could help?

Greetings, Dani

Hi Nikoleta,

which machine are you using?  We've seen similar results sometimes with our old ABI7000 with the baseline calculation set to 'auto'.  If your machine is automatically removing the background signal, try doing it manually (i.e. I would suggest setting it from cycle 3 to cycle 12 for your second graph, although you can tell best when the amplification starts by not using a log-scale on the Y axis).  The strange flat signal at the top then might resolve itself.

This seems to occur when you have lots of sample / amplification occurring early and the software can't always cope, so try manual background removal first (if you're not already) and then try diluting your input template (DNA?  cDNA?) by at least 10x if not more - the results will still be accurate if your traces are coming up at later cycles.

Good luck

I think what was meant was Ctf18.

Iam using 2mM HU ... Is this enough?

Thank you.

Hi,Nicolai, have you tried EdU or BrDU incorporation? In the case of post-mitotic tissue, in which nucleus does not incorporate BrDU, you may be able to detect events .

Good luck!

I worked for 3 years, during my first post doc on mitotic disruption (let’s call it this way…) in the UK. So, here is what I heard and read from people in the mitotic field.

First, no expert in the biology of mitosis would use this concept. This concept is a mixture of key mechanisms-events already well defined that happen in cells which cannot complete mitosis properly.

According to a recent review (see links below), the concept of “mitotic catastrophe” seems to put together different fate of cells (apoptosis, necrosis, senescence) occurring in mitotically-disrupted cells. Apparently, it is not a cell death mechanism per se… For the author mitotic catastrophe is cell death occurring during a mitotic arrest, death of cells that slipped out of a prolonged mitotic arrest, or senescence?

To be precise, one should define if a mitotic “disruptor” (DNA damage, G2 checkpoint or repair inhibitions, microtubule poisons…) induces a mitotic arrest. If cells die during this mitotic arrest, at this point people are quite convinced that these cells die by apoptosis because of the impairment of the pro / anti-apoptotic Bcl2 proteins balance, have a look at papers on degradation/phosphorylation of these proteins during mitotic arrest. Cells can also die by any mechanism during the following cycle after they slip out of mitosis. After this slippage, it is well known that cells are more or less aneuploid, tetraploid and delayed in G1 in a p53 / p38 –dependent manner, probably dependent on p21 / p16 as well (see links below). Read things about autophagy as well, which is also not thought to be a cell death mechanism per se as well.

There is also recent evidence of the implication of telomeres signalling in the fate of cells following mitotic disruption.

To conclude, I would say that talking about mitotic catastrophe is just an easy shortcut to define a far more complicated story; like it would be to talk about cell death without defining it…