ATM mediates repression of DNA end-degradation in an ATP-dependent manner.
Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.
Rahal
Elias A
EA
Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA.
Henricksen
Leigh A
LA
Li
Yuling
Y
Turchi
John J
JJ
Pawelczak
Katherine S
KS
Dixon
Kathleen
K
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D001260:Ataxia Telangiectasia / Q000235:genetics* / Q000473:pathology; D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000502:physiology*; D000077191:Wortmannin
D000730:Androstadienes; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D002110:Caffeine; D000255:Adenosine Triphosphate; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin