Biology:Ataxia telangiectasia and Rad3 related

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Short description: Protein kinase that detects DNA damage and halts cell division


A representation of the 3D structure of the protein myoglobin showing turquoise α-helices.
Generic protein structure example

Serine/threonine-protein kinase ATR, also known as ataxia telangiectasia and Rad3-related protein (ATR) or FRAP-related protein 1 (FRP1), is an enzyme that, in humans, is encoded by the ATR gene.[1][2] It is a large kinase of about 301.66 kDa.[3] ATR belongs to the phosphatidylinositol 3-kinase-related kinase protein family. ATR is activated in response to single strand breaks, and works with ATM to ensure genome integrity.

Function

ATR is a serine/threonine-specific protein kinase that is involved in sensing DNA damage and activating the DNA damage checkpoint, leading to cell cycle arrest in eukaryotes.[4] ATR is activated in response to persistent single-stranded DNA, which is a common intermediate formed during DNA damage detection and repair. Single-stranded DNA occurs at stalled replication forks and as an intermediate in DNA repair pathways such as nucleotide excision repair and homologous recombination repair. ATR is activated during more persistent issues with DNA damage; within cells, most DNA damage is repaired quickly and faithfully through other mechanisms. ATR works with a partner protein called ATRIP to recognize single-stranded DNA coated with RPA.[5] RPA binds specifically to ATRIP, which then recruits ATR through an ATR activating domain (AAD) on its surface. This association of ATR with RPA is how ATR specifically binds to and works on single-stranded DNA—this was proven through experiments with cells that had mutated nucleotide excision pathways. In these cells, ATR was unable to activate after UV damage, showing the need for single stranded DNA for ATR activity.[6] The acidic alpha-helix of ATRIP binds to a basic cleft in the large RPA subunit to create a site for effective ATR binding.[7] Many other proteins exist that are recruited to the cite of ssDNA that are needed for ATR activation. While RPA recruits ATRIP, the RAD9-RAD1-HUS1 (9-1-1) complex is loaded onto the DNA adjacent to the ssDNA; though ATRIP and the 9-1-1 complex are recruited independently to the site of DNA damage, they interact extensively through massive phosphorylation once colocalized.[6] The 9-1-1 complex, a ring-shaped molecule related to PCNA, allows the accumulation of ATR in a damage specific way.[7] For effective association of the 9-1-1 complex with DNA, RAD17-RFC is also needed.[6]   This complex also brings in topoisomerase binding protein 1 (TOPBP1) which binds ATR through a highly conserved AAD. TOPBP1 binding is dependent on the phosphorylation of the Ser387 residue of the RAD9 subunit of the 9-1-1 complex.[7] This is likely one of the main functions of the 9-1-1 complex within this DNA damage response. Another important protein that binds TR was identified by Haahr et al. in 2016: Ewings tumor-associated antigen 1 (ETAA1). This protein works in parallel with TOPBP1 to activate ATR through a conserved AAD. It is hypothesized that this pathway, which works independently of TOPBP1 pathway, is used to divide labor and possibly respond to differential needs within the cell.[8] It is hypothesized that one pathway may be most active when ATR is carrying out normal support for replicating cells, and the other may be active when the cell is under more extreme replicative stress.[8]

It is not just ssDNA that activates ATR, though the existence of RPA associated ssDNA is important. Instead, ATR activation is heavily dependent on the existence of all the proteins previously described, that colocalize around the site of DNA damage. An experiment where RAD9, ATRIP, and TOPBP1 were overexpressed proved that these proteins alone were enough to activate ATR in the absence of ssDNA, showing their importance in triggering this pathway.[7]

Once ATR is activated, it phosphorylates Chk1, initiating a signal transduction cascade that culminates in cell cycle arrest. It acts to activate Chk1 through a claspin intermediate which binds the two proteins together.[7] This claspin intermediate needs to be phosphorylated at two sites in order to do this job, something that can be carried out by ATR but is most likely under the control of some other kinase.[7] This response, mediated by Chk1, is essential to regulating replication within a cell; through the Chk1-CDC25 pathway, which effects levels of CDC2, this response is thought to reduce the rate of DNA synthesis in the cell and inhibit origin firing during replication.[7] In addition to its role in activating the DNA damage checkpoint, ATR is thought to function in unperturbed DNA replication.[9] The response is dependent on how much ssDNA accumulates at stalled replication forks. ATR is activated during every S phase, even in normally cycling cells, as it works to monitor replication forks to repair and stop cell cycling when needed.  This means that ATR is activated at normal, background levels within all healthy cells. There are many points in the genome that are susceptible to stalling during replication due to complex sequences of DNA or endogenous damage that occurs during the replication. In these cases, ATR works to stabilize the forks so that DNA replication can occur as it should.[7]

ATR is related to a second checkpoint-activating kinase, ATM, which is activated by double strand breaks in DNA or chromatin disruption.[10] ATR has also been shown to work on double strand breaks (DSB), acting a slower response to address the common end resections that occur in DSBs, and thus leave long strands of ssDNA (which then go on to signal ATR).[7] In this circumstance, ATM recruits ATR and they work in partnership to respond to this DNA damage.[7] They are responsible for the “slow” DNA damage response that can eventually trigger p53 in healthy cells and thus lead to cell cycle arrest or apoptosis.[6]

ATR as an essential protein

Mutations in ATR are very uncommon. The total knockout of ATR is responsible for early death of mouse embryos, showing that it is a protein with essential life functions. It is hypothesized that this could be related to its likely activity in stabilizing Okazaki fragments on the lagging strands of DNA during replication, or due to its job stabilizing stalled replication forks, which naturally occur. In this setting, ATR is essential to preventing fork collapse, which would lead to extensive double strand breakage across the genome. The accumulation of these double strand breaks could lead to cell death.[7]

Clinical significance

Mutations in ATR are responsible for Seckel syndrome, a rare human disorder that shares some characteristics with ataxia telangiectasia, which results from ATM mutation.[11]

ATR is also linked to familial cutaneous telangiectasia and cancer syndrome.[12]

Inhibitors

ATR/ChK1 inhibitors can potentiate the effect of DNA cross-linking agents such as cisplatin and nucleoside analogues such as gemcitabine.[13] The first clinical trials using inhibitors of ATR have been initiated by AstraZeneca, preferably in ATM-mutated chronic lymphocytic leukaemia (CLL), prolymphocytic leukaemia (PLL) or B-cell lymphoma patients and by Vertex Pharmaceuticals in advanced solid tumours.[14] ATR provided and exciting point for potential targeting in these solid tumors, as many tumors function through activating the DNA damage response. These tumor cells rely on pathways like ATR to reduce replicative stress within the cancerous cells that are uncontrollably dividing, and thus these same cells could be very susceptible to ATR knockout.[15] In ATR-Seckel mice, after exposure to cancer-causing agents, the damage DNA damage response pathway actually conferred resistance to tumor development (6). After many screens to identify specific ATR inhibitors, currently four made it into phase I or phase II clinical trials since 2013; these include AZD6738, M6620 (VX-970), BAY1895344[16] (Elimusertib).[17] and M4344 (VX-803) (10). These ATR inhibitors work to help the cell proceed through p53 independent apoptosis, as well as force mitotic entry that leads to mitotic catastrophe.[15]

One study by Flynn et al. found that ATR inhibitors work especially well in cancer cells which rely on the alternative lengthening of telomeres (ALT) pathway. This is due to RPA presence when ALT is being established, which recruits ATR to regulate homologous recombination. This ALT pathway was extremely fragile with ATR inhibition and thus using these inhibitors to target this pathway that keeps cancer cell immortal could provide high specificity to stubborn cancer cells.[18]

Examples include

Aging

Deficiency of ATR expression in adult mice leads to the appearance of age-related alterations such as hair graying, hair loss, kyphosis (rounded upper back), osteoporosis and thymic involution.[19] Furthermore, there are dramatic reductions with age in tissue-specific stem and progenitor cells, and exhaustion of tissue renewal and homeostatic capacity.[19] There was also an early and permanent loss of spermatogenesis. However, there was no significant increase in tumor risk.

Seckel syndrome

In humans, hypomorphic mutations (partial loss of gene function) in the ATR gene are linked to Seckel syndrome, an autosomal recessive condition characterized by proportionate dwarfism, developmental delay, marked microcephaly, dental malocclusion and thoracic kyphosis.[20] A senile or progeroid appearance has also been frequently noted in Seckel patients.[19] For many years, the mutation found in the two families first diagnosed with Seckel Syndrome were the only mutations known to cause the disease.

In 2012, Ogi and colleagues discovered multiple new mutations that also caused the disease. One form of the disease, which involved mutation in genes encoding the ATRIP partner protein, is considered more severe that the form that was first discovered.[21] This mutation led to severe microcephaly and growth delay, microtia, micrognathia, dental crowding, and skeletal issues (evidenced in unique patellar growth). Sequencing revealed that this ATRIP mutation occurred most likely due to missplicing which led to fragments of the gene without exon 2. The cells also had a nonsense mutation in exon 12 of the ATR gene which led to a truncated ATR protein. Both of these mutations resulted in lower levels of ATR and ATRIP than in wild-type cells, leading to insufficient DNA damage response and the severe form of Seckel Syndrome noted above.[21]

Researchers also found that heterozygous mutations in ATR were responsible for causing Seckel Syndrome. Two novel mutations in one copy of the ATR gene caused under-expression of both ATR and ATRIP.[21]

Homologous recombinational repair

Main page: Biology:Homologous recombination

Somatic cells of mice deficient in ATR have a decreased frequency of homologous recombination and an increased level of chromosomal damage.[22] This finding implies that ATR is required for homologous recombinational repair of endogenous DNA damage.

Drosophila mitosis and meiosis

Mei-41 is the Drosophila ortholog of ATR.[23] During mitosis in Drosophila DNA damages caused by exogenous agents are repaired by a homologous recombination process that depends on mei-41(ATR). Mutants defective in mei-41(ATR) have increased sensitivity to killing by exposure to the DNA damaging agents UV ,[24] and methyl methanesulfonate.[24][25] Deficiency of mei-41(ATR) also causes reduced spontaneous allelic recombination (crossing over) during meiosis[24] suggesting that wild-type mei-41(ATR) is employed in recombinational repair of spontaneous DNA damages during meiosis.

Interactions

Ataxia telangiectasia and Rad3-related protein has been shown to interact with:


References

  1. ↑ "cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein". Proceedings of the National Academy of Sciences of the United States of America 93 (7): 2850–2855. April 1996. doi:10.1073/pnas.93.7.2850. PMID 8610130. Bibcode: 1996PNAS...93.2850C. 
  2. ↑ "The Schizosaccharomyces pombe rad3 checkpoint gene". The EMBO Journal 15 (23): 6641–6651. December 1996. doi:10.1002/j.1460-2075.1996.tb01054.x. PMID 8978690. 
  3. ↑ "Quaternary structure of ATR and effects of ATRIP and replication protein A on its DNA binding and kinase activities". Molecular and Cellular Biology 24 (3): 1292–1300. February 2004. doi:10.1128/MCB.24.3.1292-1300.2003. PMID 14729973. 
  4. ↑ "Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints". Annual Review of Biochemistry 73 (1): 39–85. 2004. doi:10.1146/annurev.biochem.73.011303.073723. PMID 15189136. 
  5. ↑ "Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes". Science 300 (5625): 1542–1548. June 2003. doi:10.1126/science.1083430. PMID 12791985. Bibcode: 2003Sci...300.1542Z. 
  6. ↑ 6.0 6.1 6.2 6.3 The cell cycle : principles of control (2nd ed.). Oxford: Oxford University Press. 2012. ISBN 978-0-19-957716-3. OCLC 769544943. https://www.worldcat.org/oclc/769544943. 
  7. ↑ 7.00 7.01 7.02 7.03 7.04 7.05 7.06 7.07 7.08 7.09 7.10 "ATR: an essential regulator of genome integrity". Nature Reviews. Molecular Cell Biology 9 (8): 616–627. August 2008. doi:10.1038/nrm2450. PMID 18594563. 
  8. ↑ 8.0 8.1 "Activation of the ATR kinase by the RPA-binding protein ETAA1". Nature Cell Biology 18 (11): 1196–1207. November 2016. doi:10.1038/ncb3422. PMID 27723717. https://curis.ku.dk/ws/files/174864584/Activation_of_the_ATR_kinase_by_the_RPA_binding_protein_ETAA1.pdf. 
  9. ↑ "Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance". Genes & Development 17 (5): 615–628. March 2003. doi:10.1101/gad.1067403. PMID 12629044. 
  10. ↑ "DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation". Nature 421 (6922): 499–506. January 2003. doi:10.1038/nature01368. PMID 12556884. Bibcode: 2003Natur.421..499B. 
  11. ↑ "A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome". Nature Genetics 33 (4): 497–501. April 2003. doi:10.1038/ng1129. PMID 12640452. 
  12. ↑ "OMIM Entry - # 614564 - CUTANEOUS TELANGIECTASIA AND CANCER SYNDROME, FAMILIAL; FCTCS" (in en-us). https://omim.org/entry/614564. 
  13. ↑ "Complete loss of ATM function augments replication catastrophe induced by ATR inhibition and gemcitabine in pancreatic cancer models". British Journal of Cancer 123 (9): 1424–1436. October 2020. doi:10.1038/s41416-020-1016-2. PMID 32741974. 
  14. ↑ "Chemical strategies for development of ATR inhibitors". Expert Reviews in Molecular Medicine 16 (e10): e10. May 2014. doi:10.1017/erm.2014.10. PMID 24810715. 
  15. ↑ 15.0 15.1 "Targeting ATR in cancer". Nature Reviews. Cancer 18 (9): 586–595. September 2018. doi:10.1038/s41568-018-0034-3. PMID 29899559. 
  16. ↑ "The Novel ATR Inhibitor BAY 1895344 Is Efficacious as Monotherapy and Combined with DNA Damage–Inducing or Repair–Compromising Therapies in Preclinical Cancer Models". Molecular Cancer Therapeutics. https://aacrjournals.org/mct/article/19/1/26/92777/The-Novel-ATR-Inhibitor-BAY-1895344-Is-Efficacious. 
  17. ↑ Pusch, Fabian; Dorado García, Heathcliff; Xu, Robin; Gürgen, Dennis; Bei, Yi; Brueckner, Lotte; Röefzaad, Claudia; von Stebut, Jennifer et al. (2022). "Elimusertib outperforms standard of care chemotherapy in preclinical patient-derived pediatric solid tumor models". bioRxiv. doi:10.1101/2022.11.10.515290. https://www.biorxiv.org/content/10.1101/2022.11.10.515290v1. 
  18. ↑ "Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors". Science 347 (6219): 273–277. January 2015. doi:10.1126/science.1257216. PMID 25593184. Bibcode: 2015Sci...347..273F. 
  19. ↑ 19.0 19.1 19.2 "Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss". Cell Stem Cell 1 (1): 113–126. June 2007. doi:10.1016/j.stem.2007.03.002. PMID 18371340. 
  20. ↑ "The role of double-strand break repair - insights from human genetics". Nature Reviews. Genetics 7 (1): 45–54. January 2006. doi:10.1038/nrg1746. PMID 16369571. 
  21. ↑ 21.0 21.1 21.2 "Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR-ATRIP Seckel Syndrome". PLOS Genetics 8 (11): e1002945. 2012-11-08. doi:10.1371/journal.pgen.1002945. PMID 23144622. 
  22. ↑ "ATR suppresses endogenous DNA damage and allows completion of homologous recombination repair". PLOS ONE 9 (3): e91222. 2014. doi:10.1371/journal.pone.0091222. PMID 24675793. Bibcode: 2014PLoSO...991222B. 
  23. ↑ "High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis". PLOS ONE 9 (2): e89009. 2014. doi:10.1371/journal.pone.0089009. PMID 24551207. Bibcode: 2014PLoSO...989009S. 
  24. ↑ 24.0 24.1 24.2 "Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster". Proceedings of the National Academy of Sciences of the United States of America 73 (11): 4140–4144. November 1976. doi:10.1073/pnas.73.11.4140. PMID 825857. Bibcode: 1976PNAS...73.4140B. 
  25. ↑ "Effects of DNA-repair-deficient mutants on somatic and germ line mutagenesis in the UZ system in Drosophila melanogaster". Mutation Research 141 (1): 29–33. September 1984. doi:10.1016/0165-7992(84)90033-2. PMID 6090892. 
  26. ↑ 26.0 26.1 26.2 "Substrate specificities and identification of putative substrates of ATM kinase family members". The Journal of Biological Chemistry 274 (53): 37538–37543. December 1999. doi:10.1074/jbc.274.53.37538. PMID 10608806. 
  27. ↑ "Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress". Genes & Development 14 (23): 2989–3002. December 2000. doi:10.1101/gad.851000. PMID 11114888. 
  28. ↑ "Ataxia telangiectasia-related protein is involved in the phosphorylation of BRCA1 following deoxyribonucleic acid damage". Cancer Research 60 (18): 5037–5039. September 2000. PMID 11016625. 
  29. ↑ "Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites. In vivo assessment using phospho-specific antibodies". The Journal of Biological Chemistry 276 (20): 17276–17280. May 2001. doi:10.1074/jbc.M011681200. PMID 11278964. 
  30. ↑ 30.0 30.1 "Molecular association between ATR and two components of the nucleosome remodeling and deacetylating complex, HDAC2 and CHD4". Biochemistry 38 (44): 14711–14717. November 1999. doi:10.1021/bi991614n. PMID 10545197. 
  31. ↑ "MSH2 and ATR form a signaling module and regulate two branches of the damage response to DNA methylation". Proceedings of the National Academy of Sciences of the United States of America 100 (26): 15387–15392. December 2003. doi:10.1073/pnas.2536810100. PMID 14657349. Bibcode: 2003PNAS..10015387W. 
  32. ↑ "BRCA1-BARD1 complexes are required for p53Ser-15 phosphorylation and a G1/S arrest following ionizing radiation-induced DNA damage". The Journal of Biological Chemistry 279 (30): 31251–31258. July 2004. doi:10.1074/jbc.M405372200. PMID 15159397. 
  33. ↑ "ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses". Nature 411 (6840): 969–974. June 2001. doi:10.1038/35082110. PMID 11418864. Bibcode: 2001Natur.411..969B. 
  34. ↑ "Rheb binds and regulates the mTOR kinase". Current Biology 15 (8): 702–713. April 2005. doi:10.1016/j.cub.2005.02.053. PMID 15854902. 

Further reading

External links



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