ATM Functions to Address DSBs

During the span of a cell's life span, it'll be exposed to numerous DNA-damaging incidents anticipated to various substance and environmental providers. Should a cell be rendered struggling to repair the harm to its genetic material, malignancy and other maladies may develop, which may finally lead to the fatality of the organism. (SUZANNE CLANCEY). To this extent, numerous individuals diseases are related to an inability to correct DNA destruction. Among these diseases, ataxia telangiectasia could very well be one of the very most pertinent examples. It is an autosomal disease induced by inactivation the ataxia telangiectasia mutated (ATM) protein, where the disease provides as the protein's namesake. At the moment, ATM is believed to be accountable for initiating the phosphorylation influx of the DNA damage response in response to double-strand breaks (DSBs), which are often particularly damaging to the cell's genomic integrity due to the fact that they often bring about unfaithful DNA repair. These DSBs are also each unique in their composition and character, where the ends of the location of the DSB might not exactly follow the typical 5' phosphate and 3' hydroxyl DNA structure, and thus an array of mechanisms may be required to repair the rest. However, certain reductions may not lend themselves well to being restored, and therefore may be "blocked" from undergoing DSB repair and the one solution would thus be to sever off of the blocked section via nuclease action. But in order to review this phenomenon, it could require all DSBs to be similar in mother nature, which is false for most mutagenic agents.

To this end, Alvarez-Quilon and acquaintances utilized a specific medication known as etoposide, which is an anticancer medicine that is with the capacity of behaving as an inhibitor to the enzyme known as DNA topoisomerase II (TOP2) that can relax and unknot DNA molecules. However, during its system of action, it is required to complete duplex DNA through a momentary DSB created by the enzyme. Here, two subunits of the TOP2 are associated with each 5' end of any DSB via a phosphodiester connection. If this intermediate becomes stabilized, then transcription may become interrupted and thus makes the TOP2 to be degraded and leaving behind long term DSBs, each with peptide-based impediments at the 5' ends of the DNA. Thus, etoposide is capable of providing a consistent type of DSB, all of which have 5' end blockages. The only enzyme that is understood to remove this kind of blockage is tyrosyl DNA phosphodiesterase 2 (TDP2), and thus should this enzyme be harmed with a mutation, then any blockage produced is irreversible and requires nucleases to allow repair functions to carry on. Thus, two types of DSBs can be created: one with a genuine DSB without the blockage via making use of etoposide to wild-type skin cells (which have TDP2 intact), and another with blocked DSBs via etoposide treatment of TDP2-mutant skin cells.

Thus, the question the authors sought out to was how so when ATM functions to address DSBs. They hypothesized that ATM functions to rejoin clogged DSBs firmly when the ends were irreversibly blocked by the effect of etoposide on TOP2.

In order to find support for their hypothesis, numerous experiments were performed by Alvarez-Quilon and colleagues. One experiment the analysts performed was to analyze what would happen to the repair of clean or obstructed DSBs should ATM reduction occur. To do this, Alvarez-Quilon and fellow workers used mouse embryo fibroblasts (MEFs) that were subjected to an etoposide treatment, confluently halted at the G0/G1 cell progress stages, and analyzed Ser139-phosphorylated H2AX (ОH2AX) foci disappearance via immunofluorescence. Since ATM is accountable for phosphorylating H2AX, the disappearance of fluorescence suggests that ATM is not operating or present. Overall, it was noticed that there was a practically negligible difference in repair rate between cells with or without TDP2 (shown in Figure 2a). Cells with present TDP2 and broken ATM did not greatly affect DSB repair. However, when both TDP2 and ATM were destroyed, a large drop in the repair rate was known. The same results were seen when working with either an ATM substance inhibitor or when working with MEF cells from patients with ataxia telangiectasia (Body 2b and 2c). These results overall claim that ATM is indeed important for the repair of DSBs with clogged ends, as shown by the significantly lower repair rate in the cells without either TDP2 and ATM, while this was not observed in cells where one or both functioned.

Next, the experts wished to find out if ATM function on blocked DSBs has outcomes for the cell. To get this done, they monitored cell survival rate in response to various concentrations of etoposide. What they found was that cell survival had greatly lowered for cells lacking both TDP2 and ATM, as made evident by the actual fact that these were most vunerable to a lower development rate when subjected to increasing concentrations of etoposide (Shape 4a). This end result was more amplified when an ATM inhibitor was used, with a straight lower growth rate being obtained relative to cells with one or both of ATM or TDP2 present (Figure 4b). This might suggest that chemical type inhibition of ATM is worse for a cell than a deletion of the proteins all together, and that a non-functioning ATM may interfere with techniques related to cell growth and repair.

In addition to the cell development rate, the genome steadiness was also examined after etoposide treatment in MEFs with and without TDP2 health proteins which induced micronuclei formation (induced by mis-segregation of chromosomes or acentric chromosomal fragments were present and thus indicative of genome instability). In order to restrict the analysis performed and raise the regularity of the results, only binucleated skin cells that had blocked cytokinesis therefore of substance treatment were have scored (Shape 5a). Overall, the quantity of chromosomal aberrations increased when either ATM or TDP2 were removed (Body 5b). However, when both ATM and TDP2 were erased in the same cell, the amount of chromosomal aberrations became much higher in number than with just one of ATM or TDP2 missing. Overall, it could be said that these results serve to show ATM-mediated repair allows for an increased potential of skin cells to endure, as well concerning stabilize the integrity of the genome consequently of clogged DSBs.

As a whole, it can be said that this newspaper provided an important understanding in the role of ATM in DSB respite repair. The potential for this research mainly is based on its ability to improve understanding of ataxia telangiectasia, with the potential that new strategies of disease treatment may be determined. Furthermore, this paper contributed to a larger knowledge of DNA repair, which provides insights into additional mechanisms of repair in other organisms.

Furthermore, this paper had generally employed appropriate controls and experiments which upgraded the validity of the results obtained. However, one major issue with it is the fact that only one type of substance was employed in order to stimulate blocked DSBs. While it is convenient that this only possessed one known particular function (to disable TOP2), it is a possibility that etoposide has other results in the cell. Using another topoisomerase inhibitor may provide a broader picture concerning if ATM affects obstructed DSBs. Furthermore, only one type of block was detected (that of TOP2). Could other types of blocks have different requirements, perhaps ones not requiring ATM, or even being less reliant onto it? Other styles of chemicals can help create different blocks, that could then be analyzed for ATM function. Should these additional experiments be performed and again come back with the effect that ATM functions to remove blocked DSBs, this would provide increased credence to the results of this paper.


Clancy, S. (2008) DNA Damage & Repair: Mechanisms for Maintaining DNA Integrity. Aspect Education1(1):103

Nitiss, J. L. , Soans, E. , Rogojina, A. , Seth, A. & Mishina, M. Topoisomerase assays. Curr. Protoc. Pharmacol 57, 3. 3. 1-3. 3. 27 (2012).

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