Stable Brain ATM Message and Residual Kinase-Active ATM Protein in Ataxia-Telangiectasia

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA–dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.

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Correction of ATM mutations in iPS cells from two ataxia-telangiectasia patients restores DNA damage and oxidative stress responses

Human Molecular Genetics ◽

10.1093/hmg/ddaa023 ◽

2020 ◽

Vol 29 (6) ◽

pp. 990-1001 ◽

Cited By ~ 5

Author(s):

Dmitry A Ovchinnikov ◽

Sarah L Withey ◽

Hannah C Leeson ◽

U Wang Lei ◽

Ashmitha Sundarrajan ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Stress Responses ◽

Ataxia Telangiectasia ◽

Compound Heterozygous ◽

Gene Correction ◽

Atm Kinase ◽

Atm Protein ◽

Induced Apoptosis ◽

And Oxidative Stress

Abstract Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

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Biochemical characterization of the ataxia-telangiectasia mutated (ATM) protein from human cells

DNA Repair ◽

10.1016/j.dnarep.2004.03.041 ◽

2004 ◽

Vol 3 (7) ◽

pp. 753-767 ◽

Cited By ~ 56

Author(s):

Aaron A Goodarzi ◽

Susan P Lees-Miller

Keyword(s):

Ataxia Telangiectasia ◽

Biochemical Characterization ◽

Human Cells ◽

Ataxia Telangiectasia Mutated ◽

Atm Protein

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1FC3.3 The amounts of ATM protein and residual kinase activity predict the phenotype in Ataxia-Telangiectasia: A genotype – phenotype study

European Journal of Paediatric Neurology ◽

10.1016/s1090-3798(11)70055-1 ◽

2011 ◽

Vol 15 ◽

pp. S15

Author(s):

M. Verhagen

Keyword(s):

Ataxia Telangiectasia ◽

Kinase Activity ◽

Atm Protein

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Meiotic Telomere Distribution and Sertoli Cell Nuclear Architecture Are Altered in Atm- andAtm-p53-Deficient Mice

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7773-7783.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7773-7783 ◽

Cited By ~ 43

Author(s):

Harry Scherthan ◽

Martin Jerratsch ◽

Sonu Dhar ◽

Y. Alan Wang ◽

Stephen P. Goff ◽

...

Keyword(s):

Ataxia Telangiectasia ◽

Meiotic Prophase ◽

Nuclear Periphery ◽

Nuclear Architecture ◽

Gonadal Atrophy ◽

Atm Protein ◽

Genotoxic Insult ◽

Atm Signaling ◽

Deficient Mice

ABSTRACT The ataxia telangiectasia mutant (ATM) protein is an intrinsic part of the cell cycle machinery that surveys genomic integrity and responses to genotoxic insult. Individuals with ataxia telangiectasia as well as Atm −/− mice are predisposed to cancer and are infertile due to spermatogenesis disruption during first meiotic prophase. Atm −/−spermatocytes frequently display aberrant synapsis and clustered telomeres (bouquet topology). Here, we used telomere fluorescent in situ hybridization and immunofluorescence (IF) staining of SCP3 and testes-specific histone H1 (H1t) to spermatocytes of Atm- and Atm-p53-deficient mice and investigated whether gonadal atrophy in Atm-null mice is associated with stalling of telomere motility in meiotic prophase. SCP3-H1t IF revealed that mostAtm−/− p53 −/− spermatocytes degenerated during late zygotene, while a few progressed to pachytene and diplotene and some even beyond metaphase II, as indicated by the presence of a few round spermatids. InAtm−/− p53 −/− meiosis, the frequency of spermatocytes I with bouquet topology was elevated 72-fold. Bouquet spermatocytes with clustered telomeres were generally void of H1t signals, while mid-late pachytene and diploteneAtm−/− p53 −/− spermatocytes displayed expression of H1t and showed telomeres dispersed over the nuclear periphery. Thus, it appears that meiotic telomere movements occur independently of ATM signaling. Atm inactivation more likely leads to accumulation of spermatocytes I with bouquet topology by slowing progression through initial stages of first meiotic prophase and an ensuing arrest and demise of spermatocytes I. Sertoli cells (SECs), which contribute to faithful spermatogenesis, in theAtm mutants were found to frequently display numerous heterochromatin and telomere clusters—a nuclear topology which resembles that of immature SECs. However,Atm −/− SECs exhibited a mature vimentin and cytokeratin 8 intermediate filament expression signature. Upon IF with ATM antibodies, we observed ATM signals throughout the nuclei of human and mouse SECs, spermatocytes I, and haploid round spermatids. ATM but not H1t was absent from elongating spermatid nuclei. Thus, ATM appears to be removed from spermatid nuclei prior to the occurrence of DNA nicks which emanate as a consequence of nucleoprotamine formation.

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Nitric Oxide Induces Ataxia Telangiectasia Mutated (ATM) Protein-dependent γH2AX Protein Formation in Pancreatic β Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m113.531228 ◽

2014 ◽

Vol 289 (16) ◽

pp. 11454-11464 ◽

Cited By ~ 21

Author(s):

Bryndon J. Oleson ◽

Katarzyna A. Broniowska ◽

Katherine H. Schreiber ◽

Vera L. Tarakanova ◽

John A. Corbett

Keyword(s):

Nitric Oxide ◽

Ataxia Telangiectasia ◽

Ataxia Telangiectasia Mutated ◽

Β Cells ◽

Pancreatic Β Cells ◽

Atm Protein

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Ataxia-Telangiectasia genes and breast cancer risk in a French family study

Journal of Dairy Research ◽

10.1017/s0022029905001147 ◽

2005 ◽

Vol 72 (S1) ◽

pp. 73-80 ◽

Cited By ~ 8

Author(s):

Nadine Andrieu ◽

Eve Cavaciuti ◽

Anthony Laugé ◽

Katia Ossian ◽

Nicolas Janin ◽

...

Keyword(s):

Breast Cancer ◽

Ataxia Telangiectasia ◽

Screening Program ◽

Neuronal Degeneration ◽

Carrier Status ◽

X Rays ◽

Increased Risk ◽

Exogenous Factors ◽

Atm Protein ◽

Risk Of Cancer

Ataxia-telangiectasia (AT) is a rare autosomal recessive early childhood disorder, characterized by progressive neuronal degeneration, immunological deficiency, radiosensitivity and an increased risk of cancer caused in most cases by mutations in the AT-mutated gene (ATM). Epidemiological studies on AT families have shown that AT heterozygous women have an increased risk of developing breast cancer (BC). The ATM protein plays a central role in the recognition and repair of DNA double-strand breaks and the subsequent activation of cell-cycle checkpoints. Whilst AT is a rare disease, 0·5–1% of the general population are estimated to be AT mutation carriers, thus any increases in the risks of cancer associated with ATM carrier status are of public health relevance. The main results of our published studies on the risk of BC in 34 French AT families according to heterozygote status, type of ATM mutation and exogenous factors are summarized here. The risk of BC was higher in ATM heterozygous (HetATM) women and did not differ significantly according to the type of ATM mutation (missense vs truncating) carried by the AT family members but appeared associated with the position of some truncating mutations in certain binding domains of the ATM protein. The effect of exogenous factors, such as reproductive life factors and exposure to ionizing radiation, on the risk of BC according to ATM heterozygote status was assessed. There was no evidence for interaction (except for age at first full-term pregnancy). These findings does not appear to justify a separate screening program from that already available to other women with a first-degree relative affected by BC, as their risks have similar amplitude. Chest X-rays did not appear to be a risk factor for BC in our study population. More powerful studies, using data sets pooled from international sources are being set up to confirm these observations.

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