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. [5] [6] It is a large kinase of about 301.66 kDa. [7] 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.
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. [8] 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. [9] 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. [10] The acidic alpha-helix of ATRIP binds to a basic cleft in the large RPA subunit to create a site for effective ATR binding. [11] 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. [10] The 9-1-1 complex, a ring-shaped molecule related to PCNA, allows the accumulation of ATR in a damage specific way. [11] For effective association of the 9-1-1 complex with DNA, RAD17-RFC is also needed. [10] 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. [11] 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. [12] 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. [12]
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. [11]
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. [11] 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. [11] 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. [11] In addition to its role in activating the DNA damage checkpoint, ATR is thought to function in unperturbed DNA replication. [13] 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. [11]
ATR is related to a second checkpoint-activating kinase, ATM, which is activated by double strand breaks in DNA or chromatin disruption. [14] 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). [11] In this circumstance, ATM recruits ATR and they work in partnership to respond to this DNA damage. [11] 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. [10]
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. [11]
Mutations in ATR are responsible for Seckel syndrome, a rare human disorder that shares some characteristics with ataxia telangiectasia, which results from ATM mutation. [15]
ATR is also linked to familial cutaneous telangiectasia and cancer syndrome. [16]
ATR/ChK1 inhibitors can potentiate the effect of DNA cross-linking agents such as cisplatin and nucleoside analogues such as gemcitabine. [17] 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. [18] 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. [19] 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 [20] (Elimusertib). [21] 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. [19]
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. [22]
Examples include
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. [23] Furthermore, there are dramatic reductions with age in tissue-specific stem and progenitor cells, and exhaustion of tissue renewal and homeostatic capacity. [23] There was also an early and permanent loss of spermatogenesis. However, there was no significant increase in tumor risk.
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. [24] A senile or progeroid appearance has also been frequently noted in Seckel patients. [23] 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. [25] 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. [25]
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. [25]
Somatic cells of mice deficient in ATR have a decreased frequency of homologous recombination and an increased level of chromosomal damage. [26] This finding implies that ATR is required for homologous recombinational repair of endogenous DNA damage.
Mei-41 is the Drosophila ortholog of ATR. [27] 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 , [28] and methyl methanesulfonate. [28] [29] Deficiency of mei-41(ATR) also causes reduced spontaneous allelic recombination (crossing over) during meiosis [28] suggesting that wild-type mei-41(ATR) is employed in recombinational repair of spontaneous DNA damages during meiosis.
Ataxia telangiectasia and Rad3-related protein has been shown to interact with:
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in tens of thousands of individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur. This can eventually lead to malignant tumors, or cancer as per the two-hit hypothesis.
A molecular lesion or point lesion is damage to the structure of a biological molecule such as DNA, RNA, or protein. This damage may result in the reduction or absence of normal function, and in rare cases the gain of a new function. Lesions in DNA may consist of breaks or other changes in chemical structure of the helix, ultimately preventing transcription. Meanwhile, lesions in proteins consist of both broken bonds and improper folding of the amino acid chain. While many nucleic acid lesions are general across DNA and RNA, some are specific to one, such as thymine dimers being found exclusively in DNA. Several cellular repair mechanisms exist, ranging from global to specific, in order to prevent lasting damage resulting from lesions.
S phase (Synthesis phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during S-phase are tightly regulated and widely conserved.
ATM serine/threonine kinase or Ataxia-telangiectasia mutated, symbol ATM, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, oxidative stress, topoisomerase cleavage complexes, splicing intermediates, R-loops and in some cases by single-strand DNA breaks. It phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Several of these targets, including p53, CHK2, BRCA1, NBS1 and H2AX are tumor suppressors.
Werner syndrome ATP-dependent helicase, also known as DNA helicase, RecQ-like type 3, is an enzyme that in humans is encoded by the WRN gene. WRN is a member of the RecQ Helicase family. Helicase enzymes generally unwind and separate double-stranded DNA. These activities are necessary before DNA can be copied in preparation for cell division. Helicase enzymes are also critical for making a blueprint of a gene for protein production, a process called transcription. Further evidence suggests that Werner protein plays a critical role in repairing DNA. Overall, this protein helps maintain the structure and integrity of a person's DNA.
Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring only when favorable conditions are met. There are many checkpoints in the cell cycle, but the three major ones are: the G1 checkpoint, also known as the Start or restriction checkpoint or Major Checkpoint; the G2/M checkpoint; and the metaphase-to-anaphase transition, also known as the spindle checkpoint. Progression through these checkpoints is largely determined by the activation of cyclin-dependent kinases by regulatory protein subunits called cyclins, different forms of which are produced at each stage of the cell cycle to control the specific events that occur therein.
CHEK2 is a tumor suppressor gene that encodes the protein CHK2, a serine-threonine kinase. CHK2 is involved in DNA repair, cell cycle arrest or apoptosis in response to DNA damage. Mutations to the CHEK2 gene have been linked to a wide range of cancers.
Checkpoint kinase 1, commonly referred to as Chk1, is a serine/threonine-specific protein kinase that, in humans, is encoded by the CHEK1 gene. Chk1 coordinates the DNA damage response (DDR) and cell cycle checkpoint response. Activation of Chk1 results in the initiation of cell cycle checkpoints, cell cycle arrest, DNA repair and cell death to prevent damaged cells from progressing through the cell cycle.
DNA-dependent protein kinase catalytic subunit, also known as DNA-PKcs, is an enzyme that plays a crucial role in repairing DNA double-strand breaks and has a number of other DNA housekeeping functions. In humans it is encoded by the gene designated as PRKDC or XRCC7. DNA-PKcs belongs to the phosphatidylinositol 3-kinase-related kinase protein family. The DNA-Pkcs protein is a serine/threonine protein kinase consisting of a single polypeptide chain of 4,128 amino acids.
Cell cycle checkpoint control protein RAD9A is a protein that in humans is encoded by the RAD9A gene.Rad9 has been shown to induce G2 arrest in the cell cycle in response to DNA damage in yeast cells. Rad9 was originally found in budding yeast cells but a human homolog has also been found and studies have suggested that the molecular mechanisms of the S and G2 checkpoints are conserved in eukaryotes. Thus, what is found in yeast cells are likely to be similar in human cells.
Fanconi anemia group D2 protein is a protein that in humans is encoded by the FANCD2 gene. The Fanconi anemia complementation group (FANC) currently includes FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN and FANCO. Fanconi anemia proteins, including FANCD2, are an emerging therapeutic target in cancer
DNA topoisomerase 2-binding protein 1 (TOPBP1) is a scaffold protein that in humans is encoded by the TOPBP1 gene.
Mediator of DNA damage checkpoint protein 1 is a 2080 amino acid long protein that in humans is encoded by the MDC1 gene located on the short arm (p) of chromosome 6. MDC1 protein is a regulator of the Intra-S phase and the G2/M cell cycle checkpoints and recruits repair proteins to the site of DNA damage. It is involved in determining cell survival fate in association with tumor suppressor protein p53. This protein also goes by the name Nuclear Factor with BRCT Domain 1 (NFBD1).
The MRN complex is a protein complex consisting of Mre11, Rad50 and Nbs1. In eukaryotes, the MRN/X complex plays an important role in the initial processing of double-strand DNA breaks prior to repair by homologous recombination or non-homologous end joining. The MRN complex binds avidly to double-strand breaks both in vitro and in vivo and may serve to tether broken ends prior to repair by non-homologous end joining or to initiate DNA end resection prior to repair by homologous recombination. The MRN complex also participates in activating the checkpoint kinase ATM in response to DNA damage. Production of short single-strand oligonucleotides by Mre11 endonuclease activity has been implicated in ATM activation by the MRN complex.
The meiotic recombination checkpoint monitors meiotic recombination during meiosis, and blocks the entry into metaphase I if recombination is not efficiently processed.
The G2-M DNA damage checkpoint is an important cell cycle checkpoint in eukaryotic organisms that ensures that cells don't initiate mitosis until damaged or incompletely replicated DNA is sufficiently repaired. Cells with a defective G2-M checkpoint will undergo apoptosis or death after cell division if they enter the M phase before repairing their DNA. The defining biochemical feature of this checkpoint is the activation of M-phase cyclin-CDK complexes, which phosphorylate proteins that promote spindle assembly and bring the cell to metaphase.
DNA damage is an alteration in the chemical structure of DNA, such as a break in a strand of DNA, a nucleobase missing from the backbone of DNA, or a chemically changed base such as 8-OHdG. DNA damage can occur naturally or via environmental factors, but is distinctly different from mutation, although both are types of error in DNA. DNA damage is an abnormal chemical structure in DNA, while a mutation is a change in the sequence of base pairs. DNA damages cause changes in the structure of the genetic material and prevents the replication mechanism from functioning and performing properly. The DNA damage response (DDR) is a complex signal transduction pathway which recognizes when DNA is damaged and initiates the cellular response to the damage.
Penelope "Penny" Jeggo is a noted British molecular biologist, best known for her work in understanding damage to DNA. She is also known for her work with DNA gene mutations. Her interest in DNA damage has inspired her to research radiation biology and radiation therapy and how radiation affects DNA. Jeggo has more than 170 publications that pertain to DNA damage, radiation, and cancer research and has received 3 top science awards/medals for her research. Jeggo has also been a member of several organizations that pertain to radiation biology; these organizations include Committee on Medical Aspects of Radiation in the Environment (COMARE), National Institute for Radiation Science laboratory researcher, and the Multidisciplinary European Low Dose Initiative (MELODI). Jeggo is a member of these organizations, and she is also an editor for several publication journals that are related to cancer and radiation biology. Jeggo is very passionate about her research and in an interview with Fiona Watt claimed that “Although my results contributed only the tiniest smidgeon to scientific knowledge, I gained immense satisfaction from it”.
DNA replication stress refers to the state of a cell whose genome is exposed to various stresses. The events that contribute to replication stress occur during DNA replication, and can result in a stalled replication fork.
A double-strand break repair model refers to the various models of pathways that cells undertake to repair double strand-breaks (DSB). DSB repair is an important cellular process, as the accumulation of unrepaired DSB could lead to chromosomal rearrangements, tumorigenesis or even cell death. In human cells, there are two main DSB repair mechanisms: Homologous recombination (HR) and non-homologous end joining (NHEJ). HR relies on undamaged template DNA as reference to repair the DSB, resulting in the restoration of the original sequence. NHEJ modifies and ligates the damaged ends regardless of homology. In terms of DSB repair pathway choice, most mammalian cells appear to favor NHEJ rather than HR. This is because the employment of HR may lead to gene deletion or amplification in cells which contains repetitive sequences. In terms of repair models in the cell cycle, HR is only possible during the S and G2 phases, while NHEJ can occur throughout whole process. These repair pathways are all regulated by the overarching DNA damage response mechanism. Besides HR and NHEJ, there are also other repair models which exists in cells. Some are categorized under HR, such as synthesis-dependent strain annealing, break-induced replication, and single-strand annealing; while others are an entirely alternate repair model, namely, the pathway microhomology-mediated end joining (MMEJ).
