|
 
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| REVIEW ARTICLE |
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| Year : 2020 | Volume
: 10
| Issue : 2 | Page : 51-59 |
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ATM and ATR checkpoint kinase pathways: A concise review
Varsha Wagh1, Pranav Joshi1, Heena Jariyal2, Neelam Chauhan3
1 Department of QC Analytical, Intas Pharmaceutical Ltd., (Biopharma Division), Ahmedabad, Gujarat, India
2 Department of Biotechnology, NIPER-Ahmedabad, Gujarat, India
3 Department of Pharmacology, Karnavati School of Dentistry, Karnavati University, Gandhinagar, Gujarat, India
| Date of Submission | 27-Jun-2019 |
| Date of Acceptance | 16-Apr-2020 |
| Date of Web Publication | 13-May-2020 |
Correspondence Address:
Neelam Chauhan
Department of Pharmacology, Karnavati School of Dentistry, Karnavati University, 907/A, Uvarsad, Gandhinagar - 382 422, Gujarat
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/AIHB.AIHB_78_19
The ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia-mutated and Rad3-related (ATR) DNA damage repair pathways serve as the surveillance system which keeps a check on different types of DNA damages and lesions, which includes DNA single-strand breaks, DNA double-strand breaks and other aberrant structures such as arrested replication forks during replication. The ATM and ATR kinases belong to PIKK class of kinases which activate a large number of downstream mediator and effector molecules. The main classes of effector kinases activated by ATM and ATR are checkpoint kinase 2 and checkpoint kinase 1, respectively. ATR works primarily with the RAD9-RAD1-HUS1 (9-1-1) complex, whereas ATM works with the MRE11– RAD50–NBS1 complex. Together ATM and ATR kinase protects the cells' genomic integrity and prevents random mutations to be carried into their progeny.
Keywords: 9-1-1 complex, ataxia-telangiectasia-mutated and Rad3-related, ataxia-telangiectasia-mutated, checkpoint kinase 1, checkpoint kinase 2, DNA damage repair pathway, MRE11– RAD50–NBS1 complex
How to cite this article:
Wagh V, Joshi P, Jariyal H, Chauhan N. ATM and ATR checkpoint kinase pathways: A concise review. Adv Hum Biol 2020;10:51-9 |
| Introduction | |  |
DNA damage
In order to survive, each and every cell in the body has an intrinsic mechanism that helps in repairing the DNA damages and mutations caused due to several factors which include ionising radiations (ultraviolet [UV], IR, X-rays and gamma rays, etc.), cytotoxic drugs (doxorubicin and cisplatin), endogenous substrates (reactive oxygen species, free radical oxygen, etc.) and replication stress generated due to suboptimal culturing conditions produced during cellular differentiation. All these factors lead to damages in the form of mutations or generating aberrant structures in the double-stranded DNA (dsDNA) molecule such as single-stranded DNA segment (ssDNA) or double-stranded DNA breaks (DSB). These mutations and aberrant structures need to be repaired with utmost efficiency since they can generate an oncogenic potential in the cell and may also lead to organ failure, immunodeficiency in metazoan cells. An intrinsic surveillance system that is cell cycle checkpoint pathway constantly checks for these DNA damages in the genome to ensure that all the cells pass accurate copies of DNA to their progeny. The main function of these DNA damage repair (DDR) pathway is to check for these damages and simultaneously stop the progression of the cell cycle to provide sufficient time for DNA repair.[1]
Types of DNA damage
DNA damage may occur as a result of normal cellular metabolism (endogenous damage) or under the influence of various exogenous factors (exogenous damage); they can cause DNA damage either directly or indirectly. Each mutagen has a preference of damaging specific nucleotides so that they can produce recognisable patterns of mutagenesis. Endogenous sources of DNA damage include spontaneous or enzymatic conversions.[1] Thus, there are two main types of DNA damage. Damage of endogenous origin includes depurination, depyrimidination, deamination, alkylation, oxidation of nitrogenous bases, replication errors and free radical species. DNA breaks of endogenous origin consist of single-strand breaks (SSBs) and double-strand breaks (DSBs). SSBs occur as a result of disruption of the phosphodiester bond between two adjacent deoxyribose residues in the backbone of DNA. DSBs occur when phosphodiester backbone of both the strands of the same DNA helix is broken and the breakpoints come in proximity of each other so that the broken ends may become physically separated. Generation of free radicals or exposure to exogenous physical agents such as ionising radiations usually leads to the generation of double-strand breaks. Exogenous factors may cause many different types of DNA damage; some of this damage is specific to the action of exogenous agents only (e.g., dimerisation), whereas others may be caused by both endogenous and exogenous factors (e.g., alkylation, strand breaks, etc).[2] Exposure to non-ionising UV radiation is responsible for biochemical changes such as the formation of pyrimidine dimers.[3] Other exogenous chemical agents that can cause DNA damage include cisplatin (generates bulky adducts and interstrand or intrastrand crosslinks), benzo[a]pyrenes, daunorubicin and actinomycin-D; DNA alkylating agents include N-nitroso–N-methylurea, N-ethyl–N-nitrosourea, nitrogen mustards, methyl methanesulphonate and psoralens cause intercalation.
| Repair Mechanisms | | |
Ataxia-telangiectasia-mutated (ATM) checkpoint kinase 2 (Chk2) and ataxia-telangiectasia-mutated and Rad3-related (ATR) checkpoint kinase 1 (Chk1) are the major regulators of the DNA damage response. ATM and ATR are both large kinases having significant sequence homology and phosphorylating Ser or Thr residues that are followed by Gln. Double-stranded breaks lead to ATM activation, whereas ssDNAs activate ATR such as during replication stress, UV-induced DNA damage or stalled replication fork. ATR activation occurs during every S-phase of the cell cycle to regulate firing of replication origins and the repair of damaged replication fork and to prevent the premature entry of cells into mitosis. Mutations in ATM causes cancer predisposition and are found in approximately 0.5%–1% of the population. Mutations in both alleles of ATM cause neurodegenerative and cancer predisposition disorder ataxia-telangiectasia.[4] On the other hand, mutations in ATR are rare. A non-synonymous missense mutation in ATR is the first genetic mutation found in the Seckel syndrome.[5] Interference in ATR pathway cause genome instability and activation of ATR occurs by most cancer chemotherapeutics.
ATM and ATR, along with the mammalian target of rapamycin and also the DNA-dependent protein kinase (DNA-PK), belong to Class-IV phosphoinositide 3-kinase (PI3K) related kinase (PIKK) family. Their main role is to maintain genome integrity in eukaryotic cells. They are categorised separately since they involve catalytic loops of PI3K class 3. Other features include a conserved PIKK-regulatory domain (PRD), FRAP-ATM-TRRAP (FAT) carboxy-terminal (FATC) domains, FAT and PRD along with a large number of α-helical Huntingtin, elongation factor 3, protein phosphatase 2A, TOR1 (HEAT) repeat motifs at the N-terminal.[6] The C-terminus of the kinase domain has the FATC domain, whereas the N-terminus has the FAT domain. For the activation of ATM and ATR pathways, the kinase domains, namely PRD, FAT and FATC regions, are very important.[7] For the proper regulation of protein activity, the HEAT repeats undergo deformation when the protein molecule undergoes mechanical stress. The HEAT repeats for this purpose act as elastic scaffolds. Furthermore, for interaction with other intermediate molecules and for the stability of ATM and ATR during their functioning in the DDR, the HEAT repeat scaffolds at the N-terminal are of core importance.[8],[9] Usually, most of the members of the PI3K family exist as homodimers or heterodimers in their inactive. For their stability and reliable kinase activity, dimerisation is a prerequisite. Similarly, ATM under non-stress conditions exists as dimers or oligomer, whereas under DNA damage conditions, the oligomers dissociate to generate active monomeric forms.[7] In contrast, ATR forms a complex with the ATR-interacting protein (ATRIP); this complex formed increases the stability of ATR and is also required for the activity of ATR by interaction with replication protein A (RPA) at the replication stress site, and although the interaction with ATRIP is not a strong interaction, it is indispensable for its activity.[10],[11]
Ataxia-telangiectasia is an autosomal recessive disorder that occurs due to mutations in the genes encoding ATM and ATR.[5],[12] Ataxia-telangiectasia is a clinically incurable disorder that includes progressive ataxia, telangiectasia (spider veins), immune defects, genome instability and malignancy, whereas Seckel syndrome was characterised by postnatal dwarfism, microcephaly, intrauterine growth defects and mental retardation. Phosphorylation of proteins that contain Ser or Thr residue that is followed by Gln (SQ or TQ motifs) in ATM and ATR leads to their activation.[7] ATM activates its downstream kinase checkpoint kinase 2, whereas ATR activates its downstream kinase that is Chk1 and Chk2, encoded by CHEK1 and CHEK2, respectively. Upregulation of cell cycle checkpoint pathways occurs as a result of activation inducing cell cycle arrest and DNA repair. While ATM and the MRE11–RAD50–NBS1 (MRN) complex always work in simultaneous coordination, ATR works with TopBp1, Claspin and Rad9-Rad1-Hus1 (9-1-1) complex in the effector case along with Gamma H2AX.[13],[14]
| Atm Activation | | |
ATM exists as a multi-subunit complex (a homodimer or a higher chain of ATM monomers) in the absence of replication stress or DNA damages, and just like all the other PIKK, it needs a stimulation in the form of stress or DNA damage for its activation.[7] Therefore, the first step in ATM activation is the formation of the ATM monomers, followed by the activation of ATM-MRN complex signalling cascade. It is still unclear as to how the activators of ATM initiate the activation and recruitment of ATM at the sites of DSB's ATM undergoes autophosphorylation in its FAT domain followed by replication stress and DNA damage at the residues S1981, S367 and S1893.[15] This leads to the dissociation of ATM homodimers into its active monomeric forms.[7] The autophosphorylation also helps stabilise ATM at the damage sites.[16] Activation following induction of DNA damage in thein vivo murine models does not get affected after spontaneous mutagenesis in the residues S367, S1899 and S1987.[17] Studies suggest that the activation of ATM via autophosphorylation is not conserved across species and is dispensable for activation, whereas the monomerisation of the ATM proteins appears to be indispensable for its activation. Following DNA damage, acetylation at K3016 seems to be an important step in the autophosphorylation and monomerisation of ATM.[18] The acetylation of ATM for activation is mediated by the association of two main effectors, namely histone acetyltransferase KAT5 and histone H3 trimethylated at Lys9 (H3K9 me3), induced by phosphorylation of KAT5 by cellular kinases©.[19] The MRN complex is an important sensor for the activation of ATM. It also acts as a platform for ATM by binding to the dsDNA molecule and recruiting ATM.[20] Special features of the MRN complex which are activated upon its binding to the DNA molecule, such as conformational changes, exonuclease activity and endonuclease activity, DNA binding capability serves as an activating stimuli for ATM activation.[21],[22],[23],[24] The interaction of NBS1 which is a component of MRN complex with ATM appears to be important for the activation of the latter.[25] Although for ATM activation the MRE11 subunit of MRN complex is dispensable, the RAD50 subunit consisting of the zinc hook and the coiled domain helps in the ATM activation along with the ATP-dependant conformational changes that allow the positioning of the MRN complex at the damage sites.[26] Although the exact mechanisms by which ATM is activated by MRN complex in DNA double-stranded breaks are yet to be determined, the activity of the MRN complex appears to be of core importance in ATM-activated DDR.[27] Certain other effectors such as PP5, Wip1 and PP1 also help in ATM activation by dephosphorylating the molecule.[28],[29],[30]
| Atr Activation | | |
The ATR–ATRIP complex is loaded onto the DNA molecule along with TopBp1, which further coordinates cell cycle progression with DNA metabolic processes. ATR helps in the stabilisation of stalled replication forks and restarting fork arrest following replication stress. Whereas ATR inhibition results in enhancement in the firing of origins when there is absence of DNA damage, whereas, in replication stress, ATR phosphorylates the “Fanconianemia, complementation group I (FANC1)” and inhibits the firing of dormant origins.[31],[32] Furthermore, ATR deficiency in arrested cells causes incomplete replication of regions with fragile sites.[33],[34] When a replication fork encounters and undergoing gene transcription, the Mec1/ATR pathway is responsible for the phosphorylation of nucleoporin components and to release the transcribed chromatin that is attached to the nuclear membrane, thus preventing torsional stress-induced fork reversal for an error-free replication through different means such as by preventing fragile site expression, stabilisation of arrested replication forks, by influencing firing of origins of replication, by simultaneous coordination between replication and transcription and finally by triggering replication stress signals.[31],[32],[33],[34],[35],[36],[37] A balance between the amounts of RPA and ssDNA appears to be crucial for the stability of the replication fork during fork arrest.[38] Furthermore, there is a generation of DSBs in the molecule when there is uncoating of ssDNA by RPA molecules during fork arrest.[38]
| Atr-Chk1 Activation | | |
The nucleofilaments that are formed between RPA and ssDNA segments are responsible for the activation of the ATR-ATRIP heterodimer complex.[39] The ATR-ATRIP heterodimer interaction with RPA is an important step and a prerequisite in the localisation of activated ATR-TRIP complex at DNA damage site and at arrested replication forks, but still some studies suggest that RPA is dispensable for ATR activation and the following Chk1 phosphorylation following genotoxic stress since ATR activation also seems to take place in the absence of RPA nucleofilaments.[39],[40],[41] ATR activation begins by ubiquitylation of RPA by the pre-mRNA processing factor 19 at the damaged site.[42],[43] The next step involves the interaction of RPA-bound ATR-ATRIP complexes with the clamp loader RAD9-Rad1-Hus1 (9-1-1) complex which is bound to both ssDNA segments and dsDNA.[44] Another clamp loader complex, RAD17–RFC, which is facilitated by the activated RPA, is required to load the 9-1-1 complex onto the ssDNA–dsDNA junction.[39],[44] Finally, ATR activation occurs by the association of DNA topoisomerase 2-binding protein 1 (TopBP1) with the FATC domain of ATR after the phosphorylation of RAD9 on residue S387, which is a subunit of the 9-1-1 complex.[45],[46] Furthermore, the Rad9, Rad1, Hus1-interacting nuclear orphan protein, encoded by RHNO1, also mediates the interaction of TopBP1 which allows the interaction with both phosphorylated and unphosphorylated forms of the 9-1-1 complex. Although RHINO1 is dispensable for ATR activation, it is activated by genotoxic stress.[47],[48],[49],[50],[51] Furthermore, the initiation checkpoint signalling can occur in the absence of DNA damage by the activation of ATR-activation domain in topoisomerase by other ATR activators.[52] Some cells of phenotypes that are depleted in the RAD9 and HUS1 subunits of the 9-1-1 complex do not fully match the cells with defective ATR activation mechanism, which suggests that some of the ATR functions occur without the 9-1-1 complex.[53],[54]
| Atr in Cell Cycle Regulation | | |
Mechanisms of repair of DSBs, nucleotide excision repair and interstrand crosslink repair are heavily dependent on ATR activation.[55] Chk1 is an effector that is activated by ATR phosphorylation at S317 and S345 residues.[56],[57] Claspin binding to Chk1 by the 9-1-1 complex and RHINO is required for the activation.[51],[58],[59],[60] For assistance in replication and origin firing, chk1 activation is required as it stabilises the chromatin-bound CDC7–DBF4 apoptosis signal-regulating kinase complex.[61] In addition, regulation of cyclin-dependent kinases (CDKs) and phosphorylation of CDC25 by Chk1 also has an effect on S-phase progression.[62] The ATR-mediated Chk1 pathway prevents cells from entering mitosis with unreplicated or damaged DNA, which may lead to replication of mutated DNA and also may cause replication fork arrest, generating ssDNA segments.[63] CDK1 is maintained in its inactive state that causes G2/M phase arrest. Chk1-dependent sequestration of CDC25C into the cytoplasm and degradation of CDC25A is required for this maintenance.[62],[64],[65],[66]
| Atm in Cell Cycle Regulation | | |
ATM activation occurs mainly under oxidative stress and double-stranded DNA breaks.[20],[67],[68] The FBH1 helicase (encoded by gene FBXO18) is responsible for the recognition of damaged replication forks and it is also responsible for the activation of ATM in early DNA damage.[69] The activated ATM then initiates a large number of cellular pathways which connect cellular mechanism involved in DNA repair, metabolism, transcription and translation. Chk 2 is the most widely researched effector of ATM. ATM phosphorylates it under replication stress at residue T62.[70],[71],[72] During DSB repair, ATM phosphorylates a number of other effectors such as DNA-PKs, CtIP, RAD50, p53, MRE11, RAD9 and NBS1.[73] Chromatin relaxation and many other cellular fates such as remodelling of nucleosomes, activation of tumour suppressor p53, nuclear factor-kappaB and a number of microRNAs are dependent on ATM activation and phosphorylation of Chk 2.[74],[75],[76],[77],[78],[79],[80],[81]
| The Atm and Atr Pathway in Cell Cycle Checkpoint | | |
The cell cycle signalling functions of ATM and ATR are largely dependent on different kinases. The identification of their cellular substrates and their phosphorylation or dephosphorylation results in activation or deactivation of these substrates. These stages are of extreme importance in the DDR pathways. Studies in cells which had mutations in ATM gene hampered mainly the G1, S and G2 checkpoints. Intensive research in the area of cell cycle checkpoints has helped in the discovery and identification of many such cellular substrates for ATR and ATM. It also suggests that many of the substrates of ATM and ATR largely overlap. Thus, the functions of ATM and ATR in cell cycle checkpoints are highly crucial and they play a major role in the journey of a cell through the various stages of cell cycle, namely G1, S, G2 and M phases and their respective checkpoints.
| G1 Checkpoint in the Cell Cycle | | |
P53 protein lies at the centre of the G1 checkpoint and its accumulation is the most significant effect that occurs in the G1 checkpoint. Loss of p53 expression leads to complete G1 checkpoint abrogation although the action of p53 in the G1 checkpoint is ubiquitous.[82],[83] Most of the genotoxic agents, whether endogenous or exogenous, lead to the activation of p53 gene. This is seen by an increase in protein stability and conformation. The genotoxic agents not only activate p53 but also lead to post-transcriptional modifications which are important for their cellular function. The cyclin-dependent kinase inhibitor, p21, is one of the key targets for post-transcriptional activation by the tumour suppressor p53. The increase in p21 expression results in inhibition of cyclin A and cyclin E-associated ckd2 functions resulting in p53. This causes a block in the progression of the cell cycle from G1 to S phase of cell cycle. Tumour suppressor p53 gene undergoes heavy expression during all kinds of genotoxic stress and it activates a variety of genes, which depending on the nature of cellular damage caused by the agents either modulates the intracellular redox balance or makes the damaged cells to undergo apoptosis in cases where the damage is too extreme and cannot be repaired.[84]
The genotoxic damage-mediated post-transcriptional activation and accumulation of p53 is dependent on a large number of kinases and phosphatases along with histone acetylases and a variety of ubiquitin-conjugating enzymes.[82],[83] Studies have been done to demonstrate that A-T cells give low expression of the p53 protein upon their exposure to IR. This also helped build this correlation between ATM and p53.[85],[86] Thus, ATM has now been stated as the supreme effector of p53 phosphorylation in IR-damaged cells.[87],[88] Thein vitro phosphorylation of p53 by ATM kinases occurs at Ser 15 residue in the N-terminal of p53. The IR-induced phosphorylation of p53 protein in A-T cells is strongly suppressed and the phosphorylation of p53 at Ser 15 residue occurs rapidly and is significantly ATM dependent in the case of IR damage.[87],[89] Studies using immune complex assays showed that even ATR protein is capable of phosphorylating p53 at the Ser 15 residue in cases of acute IR-induced damages.[90],[91],[92] However, in ATR-deficient cells, the early phase of p53 phosphorylation (0–2 h) is not significantly impaired, which suggests that ATM is the major kinase which phosphorylates the Ser 15 residue of p53 during acute IR-induced cell damage.[92] However, cells which showed overexpression of ATR kinase did not show any change in their ability required to maintain the phosphorylated state of Ser 15 residue, suggesting that the maintenance of phosphorylation in later times is majorly dependent on ATR kinase. When human fibroblast cells were given UV treatment, the trigger generated an ATM-independent Ser 15 phosphorylation. The phosphorylation was strongly reduced in the later due to overexpression of ATR kinase.[92]
ATM and ATR thus have played an important role in the regulation of the phosphorylation mediated by genotoxic stress. ATM is amongst the major kinase of PIKK family which responds to IR damages and DNA damage induced by radiomimetic agents. On the other hand, ATR acts as a major PIKK kinase in genotoxic stress and it also acts as a backup in ATM-deficient cells that undergo IR damages. Furthermore, ATR is the major player in UV-induced DNA damage, which results in DNA fork stalling and subsequent replication arrest. Anti-cancer drugs (aphicolidin and cisplatin), replication stress induces (thymidine and hydroxyurea), etc., also activate ATR. Changes in the position of Ser 15 residue of p53 or any other modification in its location might trigger its dissociation from MDM2 protein, which causes the degradation, nuclear export of the protein.[93],[94] Thus, accordingly, the ATM/ATR kinases should phosphorylate Ser 15 residue and free p53 from MDM2, causing the accumulation of p53, but as it turns out, Ser 15 phosphorylation is not the only factor required for the dissociation of p53–MDM2 binding. Instead, this stimulates the p53 protein's transactivational function, thereby enhancing its binding to the transcriptional co-activator p300.[95] However, certain possibilities still exist, which suggests that p53 phosphorylation at Ser 15 allows secondary modifications in the protein and disrupts its interaction with MDM2, thereby preventing the degradation of p53.[95] Studies performed with highly purified protein samples showed that the phosphorylation of p53 at Ser 15 residue causes subsequent phosphorylation of p53 at Ser 18 by enzyme casein kinase 1. Phosphorylation at these two sites has shown to decrease the affinity between MDM2 and p53 by approximately 3 times. Although more studies are required to determine the exact conditions under which the modification caused in p53 by ATM and ATR kinases along with casein kinase 1 causes its accumulation in intact cells.
One mechanism which bolsters the hypothesis of p53 stabilisation involves another intermediate kinase called hChk2 also known as hCds1. HCds1 relays signals to p53 and other downstream regulators in ATM-expressed cells which have undergone IR damage. hChk2 is activated by ATM kinase by phosphorylation at Thr residue (Thr 68) located at the N-terminal and subsequently hChk2 causes phosphorylation of Ser 20 residue of p53 at its N-terminal.[96],[97],[98],[99],[100] The phosphorylation at Ser 20 interacts directly with the binding of p53 and MDM2 protein causing accumulation of p53 in IR-damaged cells. Mutation in hChk2 and its loss of function can lead to physiological conditions such as Li-Fraumeni syndrome which increases cancer predisposition by generating germ line mutations in p53 gene.[101]
Thus, results have now made it likely that the interaction of p53-MDM2 is one of the major ATM targets and it acts by modification in the structures of these protein substrates.[102] Structural investigations have helped in the elucidation of a carboxy-terminal motif which involves an S-Q sequence and Ser 395 residue which acts as a phosphorylation target for ATM kinase in bothin vitro and intact cells. These studies were possible because of the identification of an anti-MDM2 antibody epitope, which controlled the function of p53 degradation by MDM2. The phosphorylation of Ser 395 residue helps in stabilisation of p53, thereby interfering in the shuttling activity of MDM2 and preventing its cytoplasmic degradation.[102]
In contrast to the multiple pathways by which ATM regulates and interacts with p53 during activation of G1 checkpoints, the role of ATR during these events remains ambiguous although the ATR-chk1 pathway is activated and interacts during IR or UV-induced cellular damages. The potential role of ATR and Chk1 is huge in the S phase checkpoint.
| The S-Phase Checkpoint | | |
DNA replication process requires a high fidelity, and for this, the cellular DNA replication system should be extremely vigilant to prevent DNA structures with abnormalities from undergoing replication process. Abnormalities such as single-strand or double-strand breaks, base modifications or mismatches might interfere in the process. The checkpoint system needs to be aware of the endogenous damages such as improper base pair incorporations and stalled replication forks. Such blocks in replication process require a quick response from the cell checkpoint machinery so that the genomic integrity and stability is preserved. Homologous recombination process is the most widely undertaken repair mechanism which uses sister chromatids to precisely repair the damaged DNA during S phase.[103] In bacterial cells too in both presence and absence of genotoxic stress, cells rely heavily on homologous recombination to repair the DNA damages and also to restart the stalled replication forks.[104],[105] Apart from this, the non-homologous DNA repair mechanism also plays a role in the repair of DNA DSBs and SSBs. However, this mechanism has less accuracy and precision. Therefore, it increases the chances of inaccurately repaired DNA being carried into the M phase of cell cycle. Under ideal circumstances, the G2 phase checkpoint detects the cells with damaged DNA that enters the M phase through S phase and repairs them by utilising the cell cycle arrest.
Thus, the S phase checkpoint is the most important and skilful checkpoint. The exposure of normal cells causes a rapid response that decreases the rate of DNA synthesis due to a decrease in the replication origin firing and strand elongation.[106] Radioresistant DNA synthesis (RDS) phenotypes are generated in ATM-depleted cells that are exposed to IR rays. RDS is also induced in normal cells which show normal expression of ATM when treated with drugs that abrogate the ATM kinase activity.[92] The functions of the ATM-hChk2 pathway are not restricted only to the G1 checkpoint. Reports have shown that ATM-hChk2 pathway has downstream effector molecules in the S phase of cell cycle too. When the cells in S phase are exposed to IR rays, the ATM-hChk2 pathway is activated where is degraded the effector Cdc25A which is the tyrosine phosphatase. This prevents Cdc25A from activating the complex of cyclin A and cdk2 as the cells pass from G1 to S phase.[107] The function of cyclin A-cdk2 complex is initiating the firing of replication origins at the appropriate time during replication of DNA in the S phase. Studies done on S phase checkpoint showed that hChk2 targets the Ser 123 residue of effector Cdc25A for phosphorylation which makes the effector to undergo ubiquitin-dependent degradation. Due to the degradation of Cdc25A and subsequent inactivation of cyclinA-cdk2 complex, the firing of replication origins is suppressed. Again, RDS-like phenotypes are observed in mutant cells that have faults in their pathway right from hChk2 to cyclin A and cdk2 complex activation.[108],[109] Studies showed that the mutant hChk2 failed to phosphorylate the Cdc25A effector and were associated with the variant form of Li-Fraumeni syndrome. Furthermore, mutations in the RDS pathway increases cancer predisposition and hampers the overall genomic integrity of the cell.[103],[107]
The NBS 1 is another important effectors in S phase checkpoint. It is encoded by NBS which is mutated in human chromosomal instability disorder.[110],[111],[112] NBS and AT patients show many similar characteristics. NBS1 is an upstream regulator of Cdc25A and also shows similar phenotypes to RDS in IR-treated cells. Mre11, Rad50 and NBS1 are often found in a complex and form the maintenance complex since it plays an important role in recombinational repairs during DNA double-strand breaks. Treatment of cells with IR rays shows accumulation of activated MRN complex in the nucleus as foci close to damaged DNA strands.[113] Although the appearance of MRN complex foci is dependent on NBS1 expression, in the absence of NBS1, the Mre11-Rad50 complex performs the DNA repair functions quite capably.[112] A hypothesis suggested that these two proteins are functionally related because of their overlapping phenotype. It was validated by experiments which validated that NBS1 is phosphorylated on three serine residues Ser 343, Ser 397 and Ser 615 by ATM kinase. If any of these serine residues is substituted by alanine, the mutant NBS1 generated could not repair the defects in the NBS cells.[114],[115],[116],[117] Although the effects of phosphorylation events on the function of NBS1 are not exactly clear at present, the link between ATM and MRN complex has become more interesting with the research of hypomorphic Mre11 alleles, which causes AT-like disorders.
Recent studies have stated that ATM and ATR have been one of the critical upstream regulators in BRCA1 activation.[59] Recent evidence suggests that BRCA1 is one of the most important players in maintaining the genome integrity and replication of genome in vertebrate cells. BRCA1 also plays a role in repair mechanisms and checkpoint pathways just like the MRN complex.[118] Experiments have proved the role of BRCA1 in DNA repair mechanisms. It promotes high fidelity through homologous recombination between sister chromatids during DNA repair.[118],[119] BRCA1 is often phosphorylated by ATM and ATR kinases at multiple sites such as the C-terminal region containing around 14 SQ motifs and around 10 sites in a chain of 300 residues of amino acids (1250–1550) at the N-terminal region which are termed as SQ cluster domain (SCD).[120] In IR exposed cells, the phosphorylation of BRCA1 is reduced significantly during the absence of ATM expression, andin vitro ATM phosphorylates at least 5 sites in the SQ sites in the SCD of the five, three sites, namely Ser1387, Ser 1423, Ser 1524 are the major phosphorylation targets in IR-damaged cells.[120]
In conclusion, the S phase of cell cycle is considered to be one of the most important phases of the cell cycle. Therefore, it is of extreme importance to study the molecular machinery that acts in these mechanisms. All the proliferating cells pass through the S phase and even with their accurate DNA replication machinery; there seem to be some cells which pass on with mutated replicated DNA. These errors seem to be inevitable, and if these mutated and damaged cells remain uncorrected, may cause faulty transcription process which increases the cancer predisposition and damages the genomic integrity of the cells.
| Conclusion | | |
The research on ATM and ATR kinases has been boosted by the discovery of cDNA's encoding these proteins. These studies have helped in determining the exact mechanism by which the cell cycle surveillance systems helps in maintaining the genomic integrity and stability in a mammalian cell. Fission and budding yeasts have been at the heart of the studies that have helped in building genetic models with respect to ATM and ATR kinases. Furthermore, studies performed in Xenopus egg extracts helped in finding the distribution of function between ATM and ATR. In the partnership of ATM and ATR, ATR is considered as the housekeeping partner. In cells which have mutations in ATR genes, the damages in DNA during the replication phase, i.e., the S phase, remain unchecked, and the mutated cells get pass through the checkpoints.[121],[122] The role of ATR in genotoxic stress such as that induced by hydroxyurea and thymidine is very important and a direct link can be drafted between ATR and S phase. This is attributed to the cells which are made aware of different lesions caused by genotoxic agents by ATR when there is deoxyribonucleotide depletion or aberrant DNA structures. Apart from S phase functions, ATR also plays an important role in G1 phase checkpoint and M phase checkpoint. However, ATM is the key player in the G1 checkpoint. In contrast to ATR, ATM plays a very important role in protecting cells from a wide variety of DNA damages, specifically double strand breaks with the complexation of MRN complexes. Current researches in the field of ATM and ATR are focusing on generating specific inhibitors which can be used as potential anticancer agents in the future cancer therapies.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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