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

Meena Shrivastav, Cheryl A. Miller, Leyma P. De Haro, Stephen T. Durant, Benjamin P C Chen, David J. Chen, Jac A. Nickoloff

Research output: Contribution to journalArticle

84 Citations (Scopus)

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 ∼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.

Original languageEnglish (US)
Pages (from-to)920-929
Number of pages10
JournalDNA Repair
Volume8
Issue number8
DOIs
StatePublished - Aug 6 2009

Fingerprint

Double-Stranded DNA Breaks
Automatic teller machines
Repair
Homologous Recombination
DNA
Null Lymphocytes
Polynucleotide 5'-Hydroxyl-Kinase
Joining
Phosphorylation
Ataxia Telangiectasia Mutated Proteins
DNA-Activated Protein Kinase
Phosphotransferases
Recombinational DNA Repair
Cells
Nucleic Acid Repetitive Sequences
Mitomycin

Keywords

  • DNA repair
  • Genome stability
  • Homologous recombination

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Shrivastav, M., Miller, C. A., De Haro, L. P., Durant, S. T., Chen, B. P. C., Chen, D. J., & Nickoloff, J. A. (2009). DNA-PKcs and ATM co-regulate DNA double-strand break repair. DNA Repair, 8(8), 920-929. https://doi.org/10.1016/j.dnarep.2009.05.006

DNA-PKcs and ATM co-regulate DNA double-strand break repair. / Shrivastav, Meena; Miller, Cheryl A.; De Haro, Leyma P.; Durant, Stephen T.; Chen, Benjamin P C; Chen, David J.; Nickoloff, Jac A.

In: DNA Repair, Vol. 8, No. 8, 06.08.2009, p. 920-929.

Research output: Contribution to journalArticle

Shrivastav, M, Miller, CA, De Haro, LP, Durant, ST, Chen, BPC, Chen, DJ & Nickoloff, JA 2009, 'DNA-PKcs and ATM co-regulate DNA double-strand break repair', DNA Repair, vol. 8, no. 8, pp. 920-929. https://doi.org/10.1016/j.dnarep.2009.05.006
Shrivastav M, Miller CA, De Haro LP, Durant ST, Chen BPC, Chen DJ et al. DNA-PKcs and ATM co-regulate DNA double-strand break repair. DNA Repair. 2009 Aug 6;8(8):920-929. https://doi.org/10.1016/j.dnarep.2009.05.006
Shrivastav, Meena ; Miller, Cheryl A. ; De Haro, Leyma P. ; Durant, Stephen T. ; Chen, Benjamin P C ; Chen, David J. ; Nickoloff, Jac A. / DNA-PKcs and ATM co-regulate DNA double-strand break repair. In: DNA Repair. 2009 ; Vol. 8, No. 8. pp. 920-929.
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