Author: Gianpiero Pescarmona Date: 11/10/2007
The ATM protein was identified as the product of the gene that is mutated (lost or inactivated) in the human genetic disorder
ataxia-telangiectasia (A-T). A-T belongs to a group of human diseases that are collectively known as ‘genomic instability
syndromes’, each of which results from a defective response to a specific DNA lesion. A-T is characterized by cerebellar
degeneration, which leads to severe, progressive neuromotor dysfunction, immunodeficiency, genomic instability, thymic and
gonadal atrophy, a striking predisposition to lymphoreticular malignancies and extreme sensitivity to ionizing radiation and
DSB-inducing agents. This devastating human disorder typically combines most of the hallmarks of a defective DNA-damage
response, clearly pointing to the DSB as the DNA lesion that elicits this defect. Indeed, cultured cells from A-T patients
show a broad defect in responding to DSBs that span almost all of the known branches of this response. The striking clinical
and cellular phenotype that is caused by ATM loss clearly places this protein at a top position in the DSB-response cascade.
Ataxia Telangiectasia is an autosomal recessive disorder (1:40,000- 1:300,000); 0,35-1% heterozigotes in the general populaton.
The disorder is caracterized by defects in a number of a distnct organ systems: – progressive cerebellar
ataxia – telangiectasias in skin and conjunctiva of the eye – immunodeficency – chromosomal instability
– radiation sensitivity – increased incidence of malignancies, primarly of limphoid origin: 40%
solid tumours, 10% Hodgkin’s limphoma.
The ATM gene provides instructions for making a protein that is located primarily in the nucleus of cells, where it helps
control the rate at which cells grow and divide. This protein also plays an important role in the normal development and
activity of several body systems, including the nervous system and the immune system. Additionally, the ATM protein assists
cells in recognizing damaged or broken DNA strands. DNA can be damaged by agents such as toxic chemicals or radiation. Breaks
in DNA strands also occur naturally when chromosomes exchange genetic material during cell division. The ATM protein
coordinates DNA repair by activating enzymes that fix the broken strands. Efficient repair of damaged DNA strands helps
maintain the stability of the cell’s genetic information. Because of its central role in cell division and DNA repair, the ATM protein is of great interest in cancer research.
ATAXIA-TELANGIECTASIA – caused by mutations in the ATM gene
Researchers have identified several hundred mutations in the ATM gene that cause ataxia-telangiectasia. People with this
disorder have mutations in both copies of the ATM gene in each cell. Most of these mutations disrupt protein production,
resulting in an abnormally small, nonfunctional version of the ATM protein. Cells without any functional ATM protein are
hypersensitive to radiation and do not respond normally to DNA damage. Instead of activating DNA repair, the defective ATM
protein allows mutations to accumulate in other genes, which may cause cells to grow and divide in an uncontrolled way. This
kind of unregulated cell growth can lead to the formation of cancerous tumors. In addition, ATM mutations can allow cells to
die inappropriately, particularly affecting cells in a part of the brain involved in coordinating movements (the cerebellum).
This loss of brain cells causes the movement problems characteristic of ataxia-telangiectasia.
BREAST CANCER – associated with the ATM gene
Researchers have found that having a mutation in one copy of the ATM gene in each cell (particularly in people who have at
least one family member with ataxia-telangiectasia) is associated with an increased risk of developing breast cancer. About 1
percent of the United States population carries one mutated copy of the ATM gene in each cell. These genetic changes prevent
many of the body’s cells from correctly repairing damaged DNA. People who have only one copy of the ATM gene in each cell due to a gene deletion are also at an increased risk of developing
breast cancer. Cells that are missing one copy of the ATM gene produce half the normal amount of ATM protein. A shortage of
this protein prevents efficient repair of DNA damage, leading to the accumulation of mutations in other genes. This buildup
of mutations is likely to allow cancerous tumors to develop.
OTHER CANCERS – increased risk from variations of the ATM gene
Research suggests that people who carry one mutated copy of the ATM gene in each cell may have an increased risk of
developing several other types of cancer. In particular, some studies have shown that cancers of the stomach, bladder,
pancreas, lung, and ovaries occur more frequently in ATM mutation carriers than in people who do not carry these mutations.
The results of similar studies, however, have been conflicting. Additional research is needed to clarify which other types of
cancer, if any, are associated with ATM mutations.
Cytogenetic Location: 11q22.3
fig.1 Molecular Location on chromosome 11: base pairs 107,598,768 to 107,745,035
The maintenance of genome integrity and fidelity is essential for the proper function and survival of all organisms. This
task is particularly daunting due to constant assault on the DNA by genotoxic agents (both endogenous and exogenous),
nucleotide misincorporation during DNA replication, and the intrinsic biochemical instability of the DNA
itself.
cytotoxicity. In humans, DNA damage has been shown to be involved in a variety of genetically inherited disorders, in aging,
and in carcinogenesis.
fig.2 DNA damage
Direct Reversal The simplest of the human DNA repair pathways involves the direct reversal of the highly mutagenic alkylation
lesion O6-methylguanine (O6-mG) by the product of the MGMT gene (O6-methylguanine DNA methyltransferase).26 The O6-mG adduct
is generated in low levels by the reaction of cellular catabolites with the guanine residues in the DNA. Correction of the
lesion occurs by direct transfer of the alkyl group on guanine to a cysteine residue in the active site of MGMT in a
“suicide” reaction. The inactivated alkyl-MGMT protein is then degraded in an ATP-dependent ubiquitin proteolytic pathway.
This energetically expensive repair mechanism for the correction of a relatively simple alkyl-adduct implies O6-mG is
extremely detrimental to the cell. Accordingly, a number of chemotherapeutic agents that attack the O6 position of guanine
have been developed and are in clinical use.
methylation, deamination, or spontaneous loss of the DNA base itself.30 These alterations, although simple in nature, are
highly mutagenic and therefore represent a significant threat to genome fidelity and stability.
fig.3 BER
lesions it acts upon. The most significant of these lesions are pyrimidine dimers (cyclobutane pyrimidine dimers and 6-4
photoproducts) caused by the UV component of sunlight. Other NER substrates include bulky chemical adducts, DNA intrastrand
crosslinks, and some forms of oxidative damage. The common features of lesions recognized by the NER pathway are that they
cause both a helical distortion of the DNA duplex and a modification of the DNA chemistry.
fig.4 NER
mismatches and insertion/deletion loops (IDLs) that result from DNA polymerase misincorporation of nucleotides and template
slippage, respectively. Mispairs generated by the spontaneous deamination of 5-methylcytosine and heteroduplexes formed
following genetic recombination are also corrected via MMR. Defects in this pathway result in a so-called “mutator” cellular
phenotype characterized by elevated frequencies in spontaneous mutations and increased microsatellite instability (MSI).
Mutations in several human MMR genes cause a predisposition to hereditary nonpolyposis colorectal carcinoma (HNPCC), as well
as a variety of sporadic tumors that display MSI.
fig.5 MMR
DSB Repair Double-strand breaks (DSBs) are perhaps the most serious form of DNA damage because they pose problems for
transcription, replication, and chromosome segregation. Damage of this type is caused by a variety of sources including
exogenous agents such as ionizing radiation and certain genotoxic chemicals, endogenously generated reactive oxygen species,
replication of single-stranded DNA breaks, and mechanical stress on the chromosomes. DSBs differ from most other types of DNA
lesions in that they affect both strands of the DNA duplex and therefore prevent use of the complementary strand as a
template for repair (see BER, NER, and MMR). Failure to repair these defects can result in chromosomal instabilities leading
to dysregulated gene expression and carcinogenesis.4 To counteract the detrimental effects of these potent lesions, cells
have evolved two distinct pathways of DSB repair,45 homologous recombination (HR) and non-homologous end joining (NHEJ) . The
cellular decision as to which pathway to utilize for DSB repair is unclear, however, it appears to be largely influenced by
stage within the cell cycle at the time of damage acquisition.
HR-directed repair corrects DSB defects in an error-free manner using a mechanism that retrieves genetic information from a
homologous, undamaged DNA molecule. The majority of HR-based repair takes place in late S- and G2-phases of the cell cycle
when an undamaged sister chromatid is available for use as repair template. The RAD52 epistasis group of proteins, including
RAD50, RAD51, RAD52, RAD54, and MRE11 mediate this process. The RAD52 protein itself is thought to be the initial sensor of
the broken DNA ends. Processing of the damaged ends ensues resulting in the production of 3’ single-stranded DNA (ssDNA)
overhangs. The newly generated ssDNA ends are bound by RAD51 to form a nucleoprotein filament. Other proteins including RPA,
RAD52, RAD54, BRCA1, BRCA2, and several additional RAD51-related proteins serve as accessory factors in filament assembly and
subsequent RAD51 activities.The RAD51 nucleoprotein filament then searches the undamaged DNA on the sister chromatid for a
homologous repair template. Once the homologous DNA has been identified, the damaged DNA strand invades the undamaged DNA
duplex in a process referred to as DNA strand exchange. A DNA polymerase then extends the 3’ end of the invading strand and
subsequent ligation by DNA Ligase I yields a heteroduplexed DNA structure. This recombination intermediate is resolved and
the precise, error-free correction of the DSB is complete.
fig.6 DSB and repair
Homologous recombination (HR) takes place in late S–G2 phase and involves the generation of a single-stranded region of DNA,
followed by strand invasion, formation of a Holliday junction, DNA synthesis using the intact strand as a template, branch
migration and resolution. Non-homologous end-joining (NHEJ) takes place throughout the cell cycle and involves binding of the
KU heterodimer to double-stranded DNA ends, recruitment of DNA-PKcs (officially known as protein kinase, DNA-activated,
catalytic polypeptide (PRKDC)), processing of ends (including Artemis-dependent processing) and recruitment of the DNA ligase
IV (LIG4)–XRCC4 complex, which brings about ligation. The ataxia telangiectasia mutated (ATM) protein affects NHEJ as it is
required for Artemis-dependent end processing. ATM signalling is the main signal-transduction process that responds to a DSB.
Ataxia telangiectasia and RAD3-related (ATR) signalling is activated later after irradiation, probably when radiation-induced
lesions block replication. Both pathways lead to cell-cycle-checkpoint activation, which allows more time for repair or
permanently prevents the proliferation of damaged cells. An important step in ATM- and ATR-dependent signalling is
phosphorylation of H2A histone family, member X (H2AFX) and recruitment of the mediator proteins, mediator of DNA damage
checkpoint 1 (MDC1), tumour protein 53 binding protein 1 (TP53BP1) and the MRN (MRE11–RAD50–NBS1) complex. Additional
proteins, such as structural maintenance of chromosomes 1 (SMC1), are also recruited to the site of damage. Other proteins
that are recruited to the foci, such as replication protein A (RPA) and Bloom syndrome (BLM), have not been shown for
clarity. ATR signalling is activated by single-stranded regions of DNA that arise, for example, at stalled replication forks.
RPA coats single-stranded DNA and recruits ATR and its partner ATRIP (ATR-interacting protein). H2AFX phosphorylation,
recruitment of mediator proteins and downstream phosphorylation occur in a process that is similar to the ATM-dependent
pathway, although with some distinctions (for example, CHEK1 and CHEK2 (checkpoint 1 and 2) might be differently
phosphorylated by ATM versus ATR). HR probably depends on ATR, either to stabilize stalled replication forks and/or to
activate the process of HR. DNA crosslinks are repaired through a process that probably generates a DSB as an intermediate. b
| DDR syndromes in which pathways that respond to DSBs are affected. AT, ataxia telangiectasia; ATLD, ataxia
telangiectasia-like disorder; FA, Fanconi anaemia; FANCD2, Fanconi anaemia complementation group D2; NBS, Nijmegen breakage
syndrome; PNK, polynucleotide kinase.
fig.7 Pathways that respond to DSB
ATM is a nuclear protein kinase with a catalytic domain similar to the one of phosphatidylinositol 3-kinases (PI 3-kinases).
This protein was discovered as a mutated proteins in patients with ataxia-telagiectasia (A-T), a sever genetic disorder
characterized by cerebellar degeneration, neuromotor dysfunction, chromosomal instability, immune system defects, cancer
predisposition, and acute sensitivity to ionizing radiations.
In undamaged cells ATM is present as a dimer or ologomer molecule in which the kinase domain is silent because associated
with the FAT region of another ATM monomer. Following DSB formation, ATM rapidly autophosphorylates on residue Serine 1981,
and the inactive ATM dimers are converted (dissociated) into active ATM monomers. Active phosphorylated ATM molecules can now
interact and phosphorylate downstream proteins that affect one or more of the cell cycle checkpoints. Some of the known
substrates are the p53 protein and its ubiquitin ligase, MDM2; the Nbs1 protein; the Brca1 protein, which interacts with
other repair proteins; the checkpoint kinase 2, Chk2; the Rad17 protein and the chromatin remodeling protein SMC1. Because of
the involvement of ATM in several downstream pathways, the phosphorylation of one substrate does not provide an accurate
picture of the ATM-mediated cellular response to DNA damage. Moreover, a comparison between the signaling pathway of ATM and
those of other PI-3-like kinase domain proteins – ATR and DNA-PK – has shown no clear differentiation between the signals.
Several downstream steps are commonly shared by these molecules, suggesting a common underlying mechanism for repairing
damaged DNA.
fig.8 A model for activation of ATM by DSBs and other forms of DNA damage.
ATM resides predominantly in the nucleus in dividing cells, and responds swiftly and vigorously to DSBs by phosphorylating
numerous substrates (see below). ATM-mediated phosphorylation either enhances or represses the activity of its targets,
thereby affecting specific processes in which these proteins are involved. Similar to other active PIKKs (with the exception
of mTOR/FRAP), ATM targets serine or threonine residues followed by glutamine (the ‘SQ/TQ’ motif). The hallmark of ATM’s
response to DSBs is a rapid increase in its kinase activity immediately following DSB formation. Researchers have long been
impressed by the rapid phosphorylation of the many ATM substrates, which converts them within minutes to phosphorylated
derivatives. A marked change in the activity of ATM would account for this massive process. Initial evidence indicated that
ATM activation might involve autophosphorylation. A breakthrough in our understanding of this process came in a landmark
publication by Bakkenist and Kastan. They found that ATM molecules are inactive in undamaged cells, being held as dimers or
higher-order multimers. In this configuration, the kinase domain of each molecule is blocked by the FAT domain of the other.
Following DNA damage, each ATM molecule phosphorylates the other on a serine residue at position 1981 within the FAT domain —
a phosphorylation that releases the two molecules from each other’s grip, turning them into fully active monomers. Within
minutes after the infliction of as few as several DSBs per genome, most ATM molecules become vigorously active. Bakkenist and
Kastan35 provide evidence that the signal for ATM activation might be chromatin alterations rather than direct contact of ATM
with the broken DNA. However, it has previously been shown that, soon after damage infliction, a portion of the nuclear ATM
is recruited to DSB sites and strongly adheres to them, possibly serving as a platform for further enzymatic reactions that
take place at those sites. Both chromatin-bound and free ATM are autophosphorylated on serine 1981 (L. Moyal, unpublished
observations). So, following DSB induction, activated ATM seems to divide between two fractions: one is chromatin bound and
the other is free to move throughout the nucleus.
The list of published ATM substrates — more than a dozen at this time — is far from complete, and many ATM-dependent
responses are likely to involve ATM targets that are unknown at present. However, the study of these pathways is gradually
disclosing a remarkably broad cellular response to DSBs that is meticulously orchestrated by ATM. A close look at the network of ATM-mediated pathways that is responsible for activation of the cell-cycle checkpoints
explains how ATM precisely and decisively controls these pathways using several sophisticated strategies. The first strategy
is to approach the same effector from several different directions. A main component in the G1–S cell-cycle checkpoint is
mediated by activation and stabilization of p53, which, in turn, activates transcription of the gene that encodes the
CDK2–cyclin-E inhibitor WAF1 (also known as p21 and CIP1). The main target of ATM in this pathway is p53, which is
phosphorylated by ATM on Ser15 . This contributes primarily to enhancing the activity of p53 as a transcription factor. ATM
also phosphorylates and activates CHK2, a checkpoint kinase41, 42 that phosphorylates p53 on Ser20. This interferes with the
p53–MDM2 interaction. The oncogenic protein MDM2 is both a direct and indirect inhibitor of p53, as it serves as a ubiquitin
ligase in p53 ubiquitylation, which mediates its proteasome-mediated degradation. ATM also directly phosphorylates MDM2 on
Ser395, which interferes with nuclear export of the p53–MDM2 complex, and hence the degradation of p53 . Finally, it has been
reported that phosphorylations of p53 on Ser9 and Ser46 , and dephosphorylation of Ser376 , are ATM dependent as well,
although the function of these changes is unknown.
fig.9 ATM and related protein kinases: safeguarding genome integrity
– radiation sensitivity – increased incidence of malignancies primarly of limphoid origin: 40%
overhangs. The newly generated ssDNA ends are bound by RAD51 to form a nucleoprotein filament. Other proteins including RPA ,
RAD52, RAD54, BRCA1 , BRCA2, and several additional RAD51-related proteins serve as accessory factors in filament assembly and
catalytic polypeptide (PRKDC )), processing of ends (including Artemis-dependent processing) and recruitment of the DNA ligase
IV LIG4–XRCC4 complex , which brings about ligation. The ataxia telangiectasia mutated (ATM) protein affects NHEJ as it is
Ataxia telangiectasia and RAD3-related signalling is activated later after irradiation, probably when radiation-induced
phosphorylation of H2A histone family, member X H2AFX and recruitment of the mediator proteins, mediator of DNA damage
checkpoint 1 MDC1 , tumour protein 53 binding protein 1 (TP53BP1) and the MRN (MRE11–RAD50–NBS1) complex. Additional
proteins, such as structural maintenance of chromosomes 1 SMC1 , are also recruited to the site of damage. Other proteins
telangiectasia-like disorder; FA, Fanconi anaemia; FANCD2 , Fanconi anaemia complementation group D2; NBS, Nijmegen breakage
This protein was discovered as a mutated proteins in patients with ataxia-telagiectasia (A-T), a "sever genetic disorder":http://www.sanger.ac.uk/perl/genetics/CGP/cosmic?action=bygene&ln=ATM&start=1&end=3057&coords=AA%3AAA
Fol0lowing DNA damage, each ATM molecule phosphorylates the other on a serine residue at position 1981 within the FAT domain —
CDK2 –cyclin-E inhibitor WAF1 (also known as p21 and CIP1). The main target of ATM in this pathway is p53, which is
also phosphorylates and activates CHK2 , a checkpoint kinase41, 42 that phosphorylates p53 on Ser20. This interferes with the
