Correction of ATM mutations in iPS cells from two ataxia-telangiectasia patients restores DNA damage and oxidative stress responses.
Hum Mol Genet
; 29(6): 990-1001, 2020 04 15.
Article
em En
| MEDLINE
| ID: mdl-32037450
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Dano ao DNA
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Ataxia Telangiectasia
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Estresse Oxidativo
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Reparo do DNA
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Células-Tronco Pluripotentes Induzidas
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Proteínas Mutadas de Ataxia Telangiectasia
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Mutação
Tipo de estudo:
Etiology_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article