DNA-PKcs and ATM co-regulate DNA double-strand break repair

DNA Repair (Amst). 2009 Aug 6;8(8):920-9. doi: 10.1016/j.dnarep.2009.05.006. Epub 2009 Jun 16.

Abstract

DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and approximately 4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Ataxia Telangiectasia Mutated Proteins
  • CHO Cells
  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / metabolism*
  • Cricetinae
  • Cricetulus
  • DNA Breaks, Double-Stranded* / radiation effects
  • DNA Damage
  • DNA Repair* / radiation effects
  • DNA-Activated Protein Kinase / metabolism*
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / metabolism*
  • Enzyme Activation / radiation effects
  • Models, Biological
  • Mutation / genetics
  • Phosphorylation / radiation effects
  • Phosphothreonine / metabolism
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rad51 Recombinase / metabolism
  • Radiation, Ionizing
  • Recombination, Genetic / genetics
  • Tumor Suppressor Proteins / antagonists & inhibitors
  • Tumor Suppressor Proteins / metabolism*

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Tumor Suppressor Proteins
  • Phosphothreonine
  • Ataxia Telangiectasia Mutated Proteins
  • DNA-Activated Protein Kinase
  • Protein-Serine-Threonine Kinases
  • Rad51 Recombinase