There are a vast number of ways that cause DNA damage and the cell has an extensive network of machinery to deal with DNA damage. The DNA-damage response is a signal transduction pathway involving sensors, transducers and effectors (fig. 1)(Zhou and Elledge 2000). The effecter proteins are involved in carrying out the various activities of the DNA-damage response, including cell cycle regulation, apoptosis and DNA repair (fig. 1). Basically sensor proteins become aware of DNA damage and initiate the DNA-damage response. This leads to a number of different pathways being activated. Cell cycle checkpoints may arrest the cell cycle leading to DNA repair and a healthy cell. If this is not possible the cell death pathway or apoptosis will be activated leading to cell death, but no cancer. On the other hand if the damaged DNA is replicated cancer is very often the result. C:\Users\Rory Crowe\Desktop\the ddr.jpg
Cell cycle regulation is a vital part of the DNA-damage response. The cell cycle can be temporarily halted to give the cell the time needed to repair the damaged DNA. Throughout the cell cycle there are various checkpoints and when these checkpoints are activated the cell cycle arrests and the DNA is repaired. In a healthy organism a cell passes through the cell cycle passing its genetic material to its daughter cells. This cell cycle consists of a number of phases-G1 - S - G2 - M. There is also a Go phase where the cell is no longer dividing. The cell cycle is regulated by cyclin dependant kinases (CDKs) and cyclins. Upon receiving a pro-mitosis signal CDKs and cyclins form dimers and govern the passage of a cell through the cell cycle (Schmitt, E. and Pacquet, C. et al. 2007). Depending on the part of the cell cycle different CDKs and cyclins form dimers. The cell cycle is necessary for the fidelity of a cell and ensures the proper passage of genetic material from mother to daughter cell. Throughout the cell cycle there are a number of checkpoints so if the DNA is damaged the DNA-damage response is capable of arresting the cell cycle to allow time for the DNA to be repaired. Post-translational modifications are a very important part of the DNA-damage response as they are responsible for activating and de-activating proteins and also for targeting other proteins for degradation. In response to double-strand breaks the sensor proteins are Nbs1/Mer11/Rad50 which move to the site of DNA damage and activate ATM, the transducer protein (Schmitt and Paquet et al 2007). ATM, then in turn activates the effecter proteins, including p53, mdm3 and CHK2. These effecter proteins then carry out the various functions of the DNA-damage response. ATR is a transducer protein involved in DNA single-strand breaks which activates its effecter proteins, CHK1 and p53. As we know part of the DNA-damage response is initiating cell cycle arrest. CHK1 and CHK2 phosphorylate cdc25 leading to its ubiquitin-mediated degradation. This leads to inhibition of cdk2/cycE, arresting the cell cycle and preventing transition of the cell from G1 to S. Another target of CHK1 and CHK2 is p53, leading to its phosphorylation and stabilisation, which in turn leads to an accumulation of p21. p21 is a CDK-cyclin inhibitor which also promotes cell cycle arrest. It is much the same for G2-M progression. CHK1 and CHK2 phosphorylate cdc25a-c, arresting G2-M progression and slowing down the S phase (Zhou and Elledge 2000). There are many ways that the DNA can be damaged to initiate the DNA-damage response, including endogenous and exogenous agents. Endogenous DNA damaging agents include the likes of UV radiation, X-Rays, Gamma Rays, various cancer chemotherapy drugs, plus a number of other substances. Endogenous DNA-damaging agents are produced within the organism and include reactive oxygen species, produced from normal metabolic by-products. These DNA damaging agents are capable of causing different types of DNA damage. We have already seen mention of double-strand breaks. Along with these there are also single-strand breaks, inter-strand DNA cross links, intra-strand DNA cross links, pyrimidine dimers and nuclear type damage. As part of the DNA-damage response, the cell has ways to deal with this damage. As we have seen, upon DNA damage the cell cycle is arrested to give the cell time to repair the damage. Base excision repair repairs a single damaged base. This base is usually damaged by deamination, oxidation or alkylation. Nucleotide excision repair repairs DNA damage by UV light. This causes the formation of pyrimidine dimers. Nucleotide excision repair also repairs DNA intrastrand cross links. Miss match repair plays a role in fixing base pair miss matches and insertion/deletion loops. All these examples involve only damage to a single DNA strand so repair is quite straightforward as the other strand can be used as a template for repair. In the case of double-strand breaks, there is no template to assist in the repair of the DNA, but the cell has evolved to be able to repair double strand breaks in one of two ways; homologous recombination and non-homologous end pairing (Zhou and Elledge 2000). It is important to stress the difference between DNA damage and mutation. The mutation is a change in the sequence of the base pairs and it cannot be repaired. If a mutation occurs in one of the proteins involved in the DNA damage response the result can be catastrophic. Cancer is very often the result of one of these mutations. The DNA damage response is very important for the well-being of the organism and if DNA damage is irreversible the cell will usually initiate programmed cell death or apoptosis. This is to ensure the damaged DNA will not be passed on to daughter cells, as we shall see MIRNAS play a role in the DNA damage response by regulating various proteins that are involved in the DNA damage response.
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Fig. 2- The DDR showing all its component parts and outcomes. From: Harper, J. and Elledge, S. 2007
MicroRNA (miRNA):
MiRNA are non-coding RNAs that are about 30 nucleotides in length (Zhang, H. and Li, Y. et al 2009). They have various functions within the cell and act post-translational level to regulate gene expression by their interactions with mRNA. The first miRNA to be discovered was lin-4, which was discovered by Victor Ambrose in C. elegans (Tong A. and Nemunaitis, J. 2008). The second miRNA discovered was let-7, also in C. elegans. There have been about 900 human miRNAs discovered to date, all of which have been experimentally verified. Another 1000-2000 have been predicted using various computational software. About 30% of the miRNAs in C. elegans are conserved in mammals, and are highly conserved across the mammalian class, showing that miRNA are not only conserved between different classes but also between different species (Pothof, J. and Verkaik, N. et al 2009).
MiRNAs are initially made as large precursors known as pri-miRNAs. The pri-miRNAs are generally >1kb transcriptional products of RNA polymerase-II and contain a 5' 7-methylguanosine cap and a 3' poly-A tail (Schickel, R. and Boyerinas, B. et al 2008). The imperfect base-pairing in the pri-miRNA leads to the formation of a hairpin(stem-loop), which is recognized by Drosha (nuclear RNAse III enzyme) and Pasha (its cofactor also known as DGCR8). These two enzymes cleave the double-stranded pri-miRNA to a 70 nucleotide double-stranded RNA hairpin (stem-loop) now known as pre-miRNA with a 2 nucleotide 3' overhang (Suzuki, H. and Yamagata, K. et al 2009). This processing step occurs in the nucleus. Exportin-5 recognizes this over hang and transports the pre-miRNA into the cytoplasm, in a process requiring Ran (a GTP-binding co-factor). Now out in the cytoplasm the pre-miRNA undergoes further processing before it can become a fully functioning, mature miRNA. A 22 nucleotide double-stranded piece of RNA is excised from the pre-miRNA in a reaction involving Dicer. This double-stranded piece of RNA is referred to as a miRNA:miRNA duplex. The miRNA:miRNA duplex is then incorporated into miRNA-associated multiprotein RNA-induced silencing complex (miRISC). MiRISC contains a number of subunits including the Argonaute proteins (Ago 2) and various other proteins (TRBP and PACT). These proteins interact with the Dicer-linked miRNA:miRNA duplex within the miRISC. The duplex helix is unwound by an RNA helicase and Ago 2 cleaves and displaces one of the strands of miRNA (Schickel, R. and Boyerinas, B. et al 2008). The result is a fully functioning, mature miRNA that is part of the RISC complex. The miRNA can now carry out its various functions, including its role in the DNA-damage response. MiRNAs carry out their functions by interacting with mRNA.
The mature miRNA is able to bind to the 3' untranslated region (UTR) of the target mRNA. The function of the miRNA is twofold, degrading the target mRNA or repressing the translation of the target mRNA, depending on the type of interaction between the two. If there is perfect complementarity between the miRNA and the mRNA then the mRNA is targeted for degradation. It is the job of Ago 2, the endonucleic part of RISC, to degrade the targeted mRNA (Hermeking, H 2009). This is mainly how miRNAs interact with mRNAs in plats. If the complementarity between the miRNA and mRNA is not perfect the mRNA will not be degraded, but its translation will be repressed. This is mainly how miRNAs carry out its role in the DNA-damage response-by preventing mRNAs to be translated into such proteins that be mutated or other proteins that are capable of hindering the DNA-damage response machinery.
miRNA formation
Fig 3-The various roles of miRNAs in the DDR-translational repression or mRNA degradation. From Esquela-Kerscher, A. and Slack, F. 2006
Because the miRNAs do not need to bind perfectly with the mRNA to carry out its functions it is possible for one miRNA to regulate any number of mRNAs. It is estimated by Aurora and Slack that a single miRNA may be able to regulate up to 200 genes by their ability to bind to many different mRNAs. Potentially, therefore, about one-third of human's mRNAs could be under the control of miRNAs, but this figure is expected to increase. For example, the 3' untranslated region (UTR) of VEGF (vascular endothelial growth factor), an angiogenic factor, has 13 binding domains that different miRNAs can target such as miR-16, miR-20a and various other miRNAs (ref). this allows for the cell to have any number of mRNAs but only a certain few will be translated into protein, depending on what is needed by the cell at a particular time. because miRNAs regulate such a large amount of mRNAs misexpression of miRNAs could be detrimental to a cell and indeed to a whole organism. MRNAs have been implicated in many diseases, including cancer, as they function both as tumor suppressors and oncogenes. This is also highlighted by the fact that about 50% of all known human miRNA are located in parts of the genome known as fragile sites, which are heavily associated with cancer (Esquela-Kerscher, A. and Slack, F. 2006)
miRNA oncogenes
Fig. 4- miRNA have various roles as tumor suppressors and oncogenes. From: Esquela-Kerscher, A. and Slack, F. 2006
MicroRNAs and the DNA-damage Response:
By 2007 it was not clear what the various roles of miRNAs were. However it was known that in nearly every type of cancer there was a massive down-regulation of miRNA activity. This suggested a role for miRNAs in the DDR, as tumor suppressors. This also makes miRNAs targets for mutagenic agents as their de-activation would be advantageous to cancer cells. Since then it has been found that there are many miRNAs that have a role in the DNA-damage response. MiRNAs regulate mRNA translation preventing the formation of certain proteins and also up regulating others through their interactions with various mRNAs. As we have seen there are many proteins involved in the DNA-damage response and numerous research carried out recently have shown that miRNAs play a role in the DNA-damage response. To see what miRNAs, if any, are involved in the DDR He, L. and He, X. et al carried out miRNA expression profiles on wt and p53 deficient MEFs(mouse embryonic fibroblasts). There was one group of miRNAs whose expression was directly associated with p53 activity (fig. 5)(ref-he and he).
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Fig. 5-mir-34 family whose expression is increased in various wt cells, and their expression in p53 deficient cells of the same type. Note how the mir-34 family expression depends on p53 status. From: He, L. and He, X. et al 2007
From this the hypothesis was formed that the mir-34 may be regulated by p53. This was found to be the case by repressing p53 in MEFs, and then re-activating it. Upon re-activation of p53 there was an up-regulation of the mir-34 family (fig. 6)
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fig. 6-miR-34 family expression in MEFs. Day 0-Day 2, p53 inhibited. Day 4-Day 6, p53 re-activated. Mir-34 family levels dependent upon p53 levels. White-miR-34a, grey-miR-34b, black-miR-34c. From: He, L. and He, X. et al 2007
To further prove this theory mir-34 family levels were measured in wt and p53 deficient mice after DNA damage caused by ionizing radiation(fig. 7)
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Fig. 7-Levels of miR-34 family in untreated mice and after exposure to ionizing radiation. Note in p53 deficient mice levels of miR-34 remains very low. . White-miR-34a, grey-miR-34b, black-miR-34c.From: He, L. and He, X. et al 2007
As can be seen there was an up-regulation of mir-34 family after DNA damage in wt METs. We now know that the mir-34 family is a direct transcriptional target of p53 and mir-34 family is up-regulated by p53 in response to radiation (Kato, M. and Paranjape, T. et al 2009). As we know p53b plays a pivotal role in the DDR and causes growth arrest by arresting the cell cycle. As mir-34 family is a direct transcriptional target of p53 the role of mir-34 family was investigated further by inducing the expression of mir-34 family in IMR90 cell, which leads to inhibition of growth in the G1 phase of the cell cycle (fig. 8)(Kato, M and Paranjape, T. et al 2009).
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Fig 8- growth levels in IMR90 cells. Note how the levels of growth are highly reduced upon induction of the miR-34 family. Mir34a-circles, miR34b&c squares. From: He, L. and He, X. et al 2007
This is also found to be the case with numerous other cells including NIH-3T3, HCT-116, A549 and TOV21G (He. L, and He, X. et al 2007 ). this data shows that the mir-34 family plays a role in the DDR. This data proves that the miR-34 family plays a role in the DDR. p53 activates the miR-34 family which either blocks the translation of its targets or targets them for degradation.
We know that UV irradiation causes DNA damage in the form of intra-strand cross links. This type of damage is repaired by nucleotide excision repair. After DNA damage p53 is stabilised and causes the cell cycle to be arrested. P53, which is a tumor suppressor, has been dubbed the guardian of the genome due to the many roles it plays in the DDR. p53 is involved in cell cycle arrest, initiation of apoptosis and DNA repair. Its importance in the DDR is also highlighted by the fact that in nearly every type of cancer there is a mutation somewhere in the p53 pathway (Brosh, R. and Shalgi, R. et al 2008). After DNA damage p53 has a number of different roles that occur at different time intervals after the damage has occurred. First of all there are protein modifications which occur rapidly after DNA damage, followed by a slower response- the transcriptional reprogramming of genes. It was found that between these two responses to DNA damage miRNAs plays a role in regulating gene expression. It was suggested by lin et al that there is a link between p53 and miRNAs and subsequently miRNAs play a role in the numerous tumor suppressive pathways that p53 is heavily involved in, and therefore the DDR. The mir-34 family is made up of 3 miRNAs; mir-43a, mir-34b and mir-34c. These three miRNAs have very similar sequences (fig. 9) so are capable of carrying out very similar roles (Hermeking, H. 2009)
mir-34
Fig 9-The miR-34 family showing the homology between their sequences. From: Hermeking, H. 2009
After DNA damage the first response is by p53 which phosphorylates CDC25a, causing cell cycle arrest. The later response is when the CDC25a promoter is silenced by p21. Both of these are p53 dependant but between these two responses there was found to be a third response to DNA damage (Pothof, J. and Verkaik, N. et al 2009).
This is where miRNAs come into play in their role in the DNA-damage response. It was found that there was a down-regulation of the CDC25a mRNA before the down-regulation if the CDC25a promoter by p21. A miRNA, miR-16, was responsible for this down-regulation of CDC25a by its interaction with the CDC25a mRNA. As well as CDC25a miR-16 was also found to regulate other proteins involved in the cell cycle. It regulates cyclin D1 and cyclin E, two proteins involved in the G1 checkpoint and consequently the G1-S progression. Because of this it has been suggested that miR-16 may be a master regulator of G1-S progression (Pothof, J. and Verkaik, N. et al 2009).
This proves that miRNAs do indeed have a role in the DNA-damage response. In this case we have found that, in response to DNA-damage, miR-16 is up-regulated. MiR-16 interacts with CDC25a, cyclin D1 and cyclin E arresting the cell cycle to allow time for DNA repair, all part of the DNA-damage response. There are many other miRNAs that regulate the gene expression of many mRNAs that play a role in the DNA-damage response. A proper balance between all of these miRNAs is very important as there are some miRNAs that regulate apoptosis as well as others that regulate the cell cycle ( Zhang, H. and Li, Y. et al 2009). MiR-221 and miR-34a regulate both apoptosis and the cell cycle. There are other miRNAs that are up-regulated in response UV irradiation, along with miR-16. A study was carried out and miRNA profiling was used to see what other miRNAs were up-regulated in response to DNA damage, in this case UV irradiation. There were various miRNAs up-regulated at various time points after the induction of DNA damage. Mir-221 was up-regulated early after DNA damage and had a long lasting effect (Le Sage, C. and Nagel, R. et al 2007). We know that miR-221 regulates p27 which is involved in regulating the cell cycle. There was another family of miRNAs that were up-regulated late after the induction of DNA damage. This miRNA was the miR-34 family that we know is a p53 target. The miR-34 family of miRNAs consists of miR-34a, miR-34b and miR-34c, and it is a highly conserved family of miRNA.
p27 is involved in the DNA-damage response and cell cycle regulation. It is a CDK inhibitor and also acts a tumour suppressor. p27 has two tumour suppressive functions. 1) it reduces the rate of proliferation of cells that have already sustained DNA damage and 2) it arrests the cell cycle. In this way p27 carries out its functions as a tumour suppressor, by halting the formation of a tumour. In its role as a regulator of the cell cycle p27 also regulates the G2-M checkpoint and a decrease in the levels of p27 after DNA damage leads to increased formation of tumours. Therefore any entity that regulates p27 levels has a role in the DNA-damage response (Payne, S. and Zhang, S. et al 2008). There are two miRNAs that regulate the levels of p27, miR-221 and miR-222. p27 binds to CDK2/cycE and CDK4/cycD to arrest the cell cycle after DNA damage, as part of the DNA-damage response. An increased level of p27 arrests the cell cycle in the G1 phase and conversely a decrease in the levels of p27 leads to uninhibited cell proliferation and as a result tumourigenesis and the onset of cancer. In p27 deficient mice it was found that they are more susceptible to genotoxin induced tumours (Payne, S. and Zhang, S. et al 2008 . This shows that p27 does indeed play a role in the DNA-damage response. The two miRNAs function to down-regulate the levels of p27 in the cell. These two miRNAs can be considered oncogenes and their over-expression leads to a down-regulation of p27. Therefore these two miRNAs play a negative role in the DNA-damage response in that they act as oncogenes promoting tumourigenesis and are responsible for the down-regulation of p27, a tumour suppressor.
Many other miRNAs have also been implicated as having a role in the DDR. Mir-15 promotes apoptosis in chronic lymphocytic leukemia (CLL) by suppressing the expression of Bcl-2, an antiapoptotic protein (Noonan, EJ. and Place, RF. et al 2009)
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Fig. 10-the various miRNAs that play a role in the DDR in response to UV irradiation. From: Pothof, J. and Verkaik, S. et al 2009
