Expression of ATM in ataxia telangiectasia fibroblasts rescues defects in DNA double‐strand break repair in nuclear extracts

Abstract

Ataxia telangiectasia (A-T) is a human genetic disorder characterized by progressive cerebellar degeneration, hypersensitivity to ionizing radiation (IR), immunodeficiency, and high cancer risk. At the cellular level, IR sensitivity and increased frequency of spontaneous and IR-induced chromosomal breakage and rearrangements are the hallmarks of A-T. The ATM gene, mutated in this syndrome, has been cloned and codes for a protein sharing homology with DNA-PKcs, a protein kinase involved in DNA double-strand break (DSB) repair and DNA damage responses. The characteristics of the A-T cellular phenotypes and ATM gene suggest that ATM may play a role similar to that of DNA-PKcs in DSB repair and that there is a primary DNA repair defect in A-T cells. In the current study, the function of ATM in DNA DSB repair was evaluated in an in vitro system using two plasmids, carrying either an EcoRI-induced DSB within the lacZalpha gene or various endonuclease-induced DSB in the SupF suppressor tRNA gene. We found that the DSB repair efficiency in A-T nuclear extracts was comparable to, if not higher than, that in normal nuclear extracts. However, the repair fidelity in A-T nuclear extracts was decreased when repairing DSB with short 5' and 3' overhangs (<4 base pairs (bp)) or blunt ends, but not 5' 4-bp overhangs. Sequencing of the mutant plasmids revealed that deletions involving 1-6 nucleotide microhomologies were the major class of mutations in both A-T and normal extracts. However, the size of the deletions in plasmids from A-T nuclear extracts was larger than that from normal nuclear extracts. Expression of the ATM protein in A-T cells corrected the defect in DSB repair in A-T nuclear extracts. These results suggest that ATM plays a role in maintaining genomic stability by preventing the repair of DSB from an error-prone pathway.

ATM mediates repression of DNA end-degradation in an ATP-dependent manner

“…Such degradation probably leads to improper end-ligation and deletions which culminate in the genetic instability phenotype associated with defects in ATM. Our data is consistent with other studies indicating that the fidelity of repair rather than efficiency is primarily affected in A-T cells [4,14,15,10,16]. These studies report an elevated level of deletions and rearrangements in the repair of plasmids harboring DSBs by A-T cells or their respective extracts.…”

“…On the other hand, cells with ATM deficiencies, or A-T cells, display levels of residual un-repaired DSBs that are similar to those detected in controls [9][10][11] or at most slightly elevated [12,13]. We have previously reported comparable efficiencies of DSB repair in A-T and control nuclear extracts; however, repair in the A-T extracts resulted in a higher level of mutations, mostly deletion events [4]. These events involved rejoining at sequences of microhomology flanking a DSB.…”

“…We have previously reported a decrease in the fidelity of DSB repair in A-T nuclear extracts when compared to controls [4]. The most prominent type of mutations observed were deletion events associated with sites of microhomology flanking a break.…”

“…We have previously reported comparable DSB repair efficiencies in A-T and control nuclear extracts [4]. The fidelity of repair, however, was defective in the A-T nuclear extracts.…”

Enhancement of human cancer cell radiosensitivity by conjugated eicosapentaenoic acid - a mammalian DNA polymerase inhibitor

“…These effects are not limited to tumor cells but also affect normal cells within the tumor stroma (19,20). The cytotoxicity of radiation is mostly mediated through the generation of DNA double-strand breaks (DSBs) as demonstrated by the radiosensitivity of cells and organisms defective in the machinery of DSB repair (21)(22)(23). Ataxiatelangiectasia mutated (ATM) protein kinase is a component of these pathways and integrates the cellular response to damage by phosphorylating some key proteins involved in cell cycle regulation and DSB repair (24,25).…”